Compositions for binding to assay substrata and methods of using

ABSTRACT

Compositions and methods for binding to assay substrata in a stable and protective manner, thereby enhancing assay performance, are provided. The compositions comprise lyotropic materials (for example, lyotropic liquid and/or liquid crystalline materials) and may contain macromolecular standards, markers or capture compounds. The compositions are capable of binding to assay substrata such as that of chips that are employed for MALDI and SELDI mass spectroscopy analyzes and plates that are used for ELISA type assays.

The present application is a divisional application of U.S. patentapplication Ser. No. 11/296,696 filed Dec. 8, 2005 and claims benefit ofU.S. provisional patent application 60/634,080 filed Dec. 12, 2004, thecomplete contents of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to compositions and methods for bindingto assay substrata, including compositions containing and methodsinvolving proteins, peptides, nucleic acids and other compounds ofimportance in biochemical assays, for binding to substrata in a stable,protective, and robust manner. In particular, the invention providescompositions of lyotropic liquid and/or, preferably, liquid crystallinematerials capable of binding to assay substrata, including compositionscontaining assay-associated compounds, particularly biomacromolecules.The biomolecules of interest include molecular weight standards, diseasemarkers, and capture compounds such as antibodies, antigens, receptors,ligands, lectins, chimeras, complementary nucleic acids, antisensecompounds, avidin, etc. The lyotropic materials are capable of bindingto assay substrata, such as that of the chips that are employed forMatrix-Assisted lased Desorption Ionization (MALDI) and Surface-EnhancedLaser Desorption Ionization (SELDI) mass spectroscopy analyses,providing a stable, protective environment for the compounds and arobust means for deposition on the chip with resulting improvement insignal strength and reproducibility. They are also capable of binding tosubstrata used in more traditional types of protein assays, such asEnzyme-Linked ImmunoSorbent Assays (ELISAs), for effective deposition ofreagents and markers, as well as for blocking non-specific binding(NSB).

2. Background of the Invention

Assays based on mass spectroscopy techniques, such as Matrix-AssistedLaser Desorption Ionization (MALDI) and Surface-Enhanced LaserDesorption Ionization (SELDI) are gaining importance in a number ofanalytical applications, including early detection of cancer, infectiousdiseases, and other pathological conditions. In mass spec as well as inother assay methods, it can be important to have one or more standardspresent, added (“spiked”) to the sample fluid, in order to provide forcalibration of both the charge/mass ratio and the intensity. However, insuch applications, the presence of compounds in biological fluids thatcan degrade proteins, peptides and other standards is in many casesinevitable; such compounds include proteases, lysozyme, trypsin,nucleases, etc. Facilitating the use of simple, relatively inexpensive,and well-studied standards such as peptides and proteins calls for amethod to protect the standard molecule from degradative enzymes andother conditions or compounds, and for accomplishing a high degree ofsubstrate binding for signal enhancement.

In addition, SELDI is a mass spec technique that, through the use ofsample substrata with specially tailored surface chemistries, can be oftremendous advantage in selecting desired standards and markers as wellas increasing their signal:noise ratios, but is currently not used tofull advantage. As an example, in the case where two standard moleculesare used in order to provide better calibration, the variance in bindingbetween the two (or more) molecules on SELDI chips, the run-to-runvariability of the peak positions and intensities, and variability inthe SELDI chips themselves, confounds the calibration of peak positions(m/z ratios) and intensities. This is particularly true in cases whereimprecision in calibration of m/z ratios leads to improper integrationof peaks.

In the art of laser-desorption mass spectrometry a number of substrateshave been developed for selective adsorption of targeted molecules ofimportance in, e.g., biomedical assays. U.S. Pat. No. 6,579,719 forexample describes methods for applying charged andhydrophobic-interaction surfaces for selective capture of biomarkers inthe context of laser-desorption mass spectrometry.

In solid-phase assays, there is a need for protein-friendly, evenbiomimetic, materials and methods for hosting capture molecules andother assay-associated proteins, in such a way that the analytemolecules are captured efficiently and can be brought down to thesubstrate with high affinity. There is a fundamental challenge in thisendeavor which has placed limitations on the quantification,specificity, and ease of use of current methods, and this challenge isvery pronounced in certain cases, such as the case of receptor-basedassays: namely, by definition, solid-phase assays involve solid-liquidinterfaces that tend to denature sensitive proteins such as receptors,as well as other membrane-associated proteins. Indeed, it is well knownthat membrane-associated proteins tend to denature or flocculate overtime even in simple aqueous (buffered) solution, and the more maturetechniques in the study of these compounds ensure that at least somelipid is retained in the preparations used in analyses. The analysis ofligand-receptor interactions is of central importance in the screeningof potential pharmaceutical actives, and yet there remains a majorunsolved problem, at the time of this writing, of how to design amaterial that will preserve the natural functionality andcharacteristics of receptor proteins and other functionalbiomacromolecules and, at the same time, exhibit desired binding touseful substrata. Broadly speaking, a solid surface is an excellentmeans by which to concentrate species which, in solution or suspension,would be so dilute as to be difficult to quantify, yet the same surfacecan wreak havoc with delicate proteins such as receptors. Evenglycolipid receptors, such as bacterial adhesin receptors, have beenshown to yield erroneous, non-physiologic binding selectivity resultswhen used in traditional solid-phase assays, due to improperpresentation of the saccharide head groups when the lipid is adsorbed toa solid surface. There is a clear need, particularly in thepharmaceutical industry, for materials that can provide anear-physiologic conformation and presentation of membrane proteins andreceptors, preferably with access to both binding and active sites,yielding a degree of fidelity obtainable perhaps only with wholecell-based assays but in a simpler and more controlled system.

Regulatory feedback can alter receptor-based physiological responses,which are further contingent on interactions between different hormonalor signaling systems, and so it is important to interpret, e.g.,pharmaceutical screening studies in the context of biochemical datareflecting direct receptor effects of drugs, in purified systems freefrom extraneous components. Furthermore the need for whole, intactreceptor molecules hosted in a physiologic milieu is crucial in view ofallosteric effects, competitive binding, multisite binding,desensitization, and other effects that quantitatively and evenqualitatively modify binding. Allosteric effects, involving the globalprotein, which drive signal transduction, are in many receptors drivenby the lower free energy associated with binding site/ligand interactionafter binding-induced conformational changes; thus, in the absence ofthe entire protein and associated allosteric effects, studies ofcompetitive binding can be qualitatively incorrect. In addition, withcertain multisite receptors, it is known that the natural ligand andexogenous agonists/antagonists can bind to different sites, and so anassay based on a partially expressed protein exhibiting only the naturalligand binding site would yield false negatives with exogenouscompounds, and the opportunity afforded by the new potential drug mightwell be missed. Similarly, in receptors such as the 5-HT-2c receptor,where the binding site involves a transmembrane domain, as well as incases where the site is at the membrane/water interface or (as in then-acetylcholine receptor) at the interface between two subunits, itwould be erroneous to work only with a partially expressed proteinrepresenting a putative binding site. Discrimination between agonist andantagonist binding sites will clearly require intact receptor, and evensuch events as dimerization of the EGF receptor, which has a strongeffect on binding affinity, apparently requires intact receptors, asreceptor-related molecules such as the secreted binding domain andgp74v-erbB do not give evidence of dimerization. In view of these facts,there is a need to improve drug-screening assays by satisfying the needfor a receptor with its allosteric regulatory mechanism intact, and withproper presentation and accessibility of binding site(s).

Liposomes have been used in conjunction with various biochemical assays,but suffer from instabilities, leakage, opsonization-related problems,incompatibilities with many proteins including membrane-associatedproteins, and generally, greatly restricted access to the compounds theyencapsulate. Concerning protein incompatibilities, even insulin has beenshown to induce leakage of DPPC liposomes through bilayer interactions[Xian-rong et al., Acta Pharm. Sinica (2000) 35(12):924]. Theselimitations can preclude their use as carriers for bioactive and capturemolecules, or at least require tethering of these compounds vialaborious and/or expensive conjugation procedures. Use of liposomes indiagnostics is largely limited to the use of high-transition temperaturelipid bilayers because of their resistance to instability,rancidification, and opsonization, at least in the case of ready-to-useproducts. Obviously crystalline materials are essentially non-functionalas solvents, and thus integral proteins cannot be incorporated. This inturn largely limits the use of liposomes to the encapsulation ofcompounds inside the aqueous interior of a rigid liposome, leaving thecompound inaccessible to the crucial intermolecular interactions thatare central to, for example, immunoassays. Furthermore, the sphericalshape of liposomes is simply not conducive to intimate substratecontact.

In summary, it would be a boon for researchers, clinical chemists,pharmaceutical scientists and others dealing with bioassays to haveavailable compositions (e.g. carrier particles) whereby assay-associatedmolecules could be sequestered, protected, and subsequently depositedreliably on selected substrata.

Simple micelles and microemulsion droplets are not well suited ascarrier particles for helping to bind macromolecules to substrata, andalso poorly suited for providing a protective encapsulation. Both arevery labile, not to be viewed as having any sort of permanence, and inthe current theory are viewed as very rapidly exchanging material witheach other, with any surfaces present, and with the aqueous domains. Andif an ionic interaction between surfactant and substrate weresufficiently strong, the likely result would not be micelles ormicroemulsion droplets adsorbed to the substrate, but rather individualmolecules adsorbed (to form a monolayer, or perhaps multilayer).

In addition, one of the commonly held beliefs by those practiced in theart of MALDI has been that the presence of lipids in samples, suppressesionization and therefore is detrimental to MALDI analysis. Further,since SELDI is a form of MALDI, one might have expected that theaddition of lipids to samples would have caused the expected ionsuppression. This belief obviously has taught away from the use oflipid-based materials in connection with MALDI and SELDI.

The prior art has thus far failed to provide compositions, and methodsfor their use, whereby standard molecules can be bound to assaysubstrata, particularly in a manner whereby the standards are protectedand stabilized. Similarly, materials for hosting biospecific capturemolecules and other assay-associated compounds, and for sequesteringanalytes from solution, have suffered from a non-physiologic nature,laborious conjugation procedures, sub-optimal substrate binding,limiting instabilities, poor presentation of binding groups, and/orobstructions to key molecular interactions.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for binding toassay substrata, including compositions containing biomolecules such asstandards, disease markers, and capture compounds. The compositions canbe bound to assay substrata, such as the surface of an assay chip orother support. The compositions comprise lyotropic materials (such aslyotropic liquids or liquid crystalline materials, and in particularcubic phase materials), and the standards and/or capture compounds arecontained within the lyotropic material. The invention is based on thediscovery that such compositions stably bind to surfaces such as thoseused for the substrata of many assay systems, e.g. to the surface ofSELDI-MS chips and ELISA plates. Not only do the compositions bind tosuch surfaces, they do so in a manner that retains the standard orcapture (and captured) molecule(s) within the protective, stableenvironment of the lyotropic material. As a result, the standards andcapture compounds are not exposed to the many potentially harmfulsubstances that are present in biological samples nor to the denaturingeffects of certain surfaces or environments, their integrity ispreserved, and the accuracy of measurements relying on these compoundsis enhanced. Surprisingly, it has also been found that at least some ofthese compositions and methods substantially or even dramatically reducethe run-to-run variability of laser-desorption mass spec measurementsand increase signal:noise ratios.

One aspect of the instant invention is the binding of lyotropic liquidor, more preferably, liquid crystalline material, to a substrate so asto coat (partially or fully) the substrate with either a collection ofparticles or a film, which in turn may or may not be coated. In somecases, the lyotropic material and the substrate will be chosen together,in tandem, so as to yield the desired binding. This can be accomplishedby judicious use of one or more of the following three generalapproaches:

A) coating: particles of lyotropic material are at least partiallycovered with a coating material that is selected so as to bind to thesubstrate;

B) compound in the lyotropic material: the lyotropic material is chosenso as to incorporate one or more compounds that promote binding of thematerial to the substrate; most preferably these compounds arebilayer-associated; less preferably, a non-bilayer-associated compoundin the lyotropic material is retained in the material by a gelation stepthat is carried out within the lyotropic material;

C) hydrophobic interaction: the lyotropic material and substrate areselected in such a way that a hydrophobic interaction between the twopromotes binding.

It is an exemplary embodiment of this invention to provide compositionsincorporating proteins and/or peptides, dendrimers or othermacromolecules, wherein said compositions bind to MALDI and SELDIsubstrata and other assay substrata, including especially compositionscomprising microparticles and coated microparticles of nanostructuredliquid and liquid crystalline phase materials. Such compositions canprovide protection of the standard molecule by encapsulation, or byincorporation in a matrix that is not easily penetrated by degradativeenzymes. They can be used to standardize charge:mass ratios, as well asintensities, in MALDI and SELDI measurements, thereby yielding greateraccuracy and enhanced capabilities. Virtually any number of peptides orproteins, for standardization or specific capture, can be incorporatedinto a system of particles with much greater control over the resultingmolar ratios between the various proteins on the assay chip, sinceentire particles can be bound to the chip along with their full payload.In one embodiment of the invention, capture molecules (e.g., antibodies,receptors, etc.) are incorporated in or at the surface of such lyotropicmicroparticles, particularly those based on reversed liquid crystallinephase materials and most preferably on reversed cubic phase materials,allowing specific capture of important analyte molecules. This attributecan be coupled with the strongly substrate-binding property of thecompositions described herein, to yield a synergistic combination ofselective analyte capture and substrate deposition. In yet anotherembodiment, the compositions can be used as blocking agents inimmunoassays and related assay methods, to limit non-specific binding(NSB) and increase sensitivity and accuracy.

In preferred embodiments, such a composition includes one or moreparticles comprising a matrix consisting essentially of a nanostructuredliquid or liquid crystalline phase material, most preferably a reversedlyotropic liquid crystalline material. Such a particle achieves itsbinding to a selected substrate by virtue of a preselected surfacechemistry, which can be, for example, cationic charge, anionic charge,hydrophobicity, chelating groups, hydrogen bonding groups,avidin/biotinylation, and the presence of antibodies, lectins, nucleicacids, receptors, chimera, and other biospecific targets at or near thesurface of the particle. In the preferred embodiments, this surfacechemistry can be attained either on a coated particle of nanostructuredliquid or liquid crystalline phase interior, or at the surface of anuncoated particle most preferably of a reversed liquid crystallinephase. The particles can likewise comprise chemical moieties that bindto specific antigens or other molecules to be captured, such as specificantigens in the body that are most preferably markers of disease. Thus,capture molecules such as antibodies and the associatedcapture-promoting interactions (e.g., antigen-antibody interaction) canplay two rather different roles in these particles: as a means tosequester analyte molecules from solution prior to substrate binding,and as a means to achieve binding to a substrate incorporating theappropriate compound. A particularly instructive example of this, dualfunctionality is the following: a lyotropic liquid crystalline particlecontaining a first antibody to an analyte binds the analyte fromsolution (e.g., from diluted serum), and the particle in turn binds to asecondary antibody to the same antigen immobilized at the substratesurface.

It is another exemplary embodiment of this invention to provide methodsfor producing and using such particles. In particular, a preferredmethod of using such particles (illustrated schematically in FIG. 1)comprises addition of the particles containing one or more of saidcompounds to a sample of biological material, such as serum, incubationof the now-spiked serum with the appropriate SELDI substrate, washingaway of non-attached material, and subsequently applying a laserenergy-absorbing matrix and performing SELDI-MS as per normal operation;the mass spectrometry peaks recorded from the encapsulatedmacromolecules then provide an accurate standardization of thecharge:mass ratio (known from the MW of the macromolecule, which isselected to be readily distinguishable from expected endogenousmacromolecules, and exhibiting sharp, well-defined MS peaks), and of theintensities provided that intensities, from the encapsulated markermacromolecules are reproducible to sufficient accuracy. Preferably,compositions that comprise everything needed to make this procedure workin a turn-key fashion are used. Preferably, the particles are in astabilized form that is compatible with the biological material, andcontain one or more macromolecules (e.g. peptides, proteins, etc.) suchthat the mass spec signal intensity from the use of the composition issignificantly greater than the signal which would be obtained with thesame amount of macromolecule in the absence of the particles, forexample with an aqueous solution of the macromolecule. Certain preferredparticles of the invention have the property that they comprise capturemoieties, such as antibodies and the like, and will carry capturedmolecules down to the desired surface upon binding to that surface, beit a SELDI substrate (illustrated schematically in FIG. 2 b) or otherassay substrate (FIG. 2 a); thus the method of using comprisescontacting the particles with a biological solution possibly containingthe molecule or antigen to be captured, and at some point before, after,or during that time, contacting the particles or a dispersion thereofwith the substrate of interest.

In another exemplary embodiment (illustrated in FIG. 3), certainpreferred, particles enhance surface binding of a molecule or antigen tobe studied in a sample even without the incorporation of a specificcapture molecule in the particles, by contacting the particles with abiological solution containing sample and at some point before, after,or during that time, contacting the particles or a dispersion thereofand sample solution with the substrate. (FIG. 3). This may enhancematrix deposition on the substrate in assays such as MALDI and SELDI,for example by inducing a much finer and more uniform deposition of theenergy-absorbing matrix and/or a more intimate association between itand the analyte material.

In yet another exemplary embodiment (schematically illustrated in FIG.4), similar particles are applied to a substrate such as an ELISA platein order to block areas where non-specific binding can otherwise occur.

The invention provides a calibration method for use in assays,comprising the steps of: 1) applying to a surface of a substrate acomposition comprising lyotropic liquid or liquid crystalline materialin which is incorporated one or more marker molecules, said compositionbinding to the surface of said substrate; and 2) using data which isderived from said one or more marker molecules as a calibrationstandard. In one embodiment, the composition is provided in the form ofparticles, which may be coated or uncoated. Two different markermolecules may be present in two different particles, or in the sameparticle. In one embodiment, the one or more marker molecules areproteins or peptides, for example, proteins and peptides that areapplicable to cancer detection. Relevant assays include ELISA assays,MALDI assays and SELDI assays. In some embodiments, the binding may bevia, for example, hydrogen bonding or ionic bonding. In one embodiment,the substrate is inorganic. In some embodiments, the composition isprovided in the form of a film. In some embodiments, the lyotropicliquid or liquid crystalline material is cubic phase.

The invention further provides a composition or kit used for calibrationin an assay. The composition or kit comprises 1) at least a firstparticle formed from a lyotropic liquid or liquid crystalline materialand having a first protein or peptide marker molecule; and at least asecond particle formed from a lyotropic liquid or liquid crystallinematerial and having a second protein or peptide marker molecule, whereinsaid second protein or peptide marker molecule is different from saidfirst protein or peptide marker molecule, and wherein each of said atleast a first particle and said at least a second particle bind directlyto a surface of a substrate suitable for use in an assay. In someembodiments, the at least a first particle and said at least a secondparticle are coated; in others, they are uncoated. The at least a firstparticle and said at least a second particle may be combined in a singlecontainer. Alternatively, they may be stored in separate containers. Inone embodiment, the substrate is used in an assay such as, for example,an ELISA, MALDI, or SELDI assay.

The invention further provides a method of performing an assay for amolecule of interest in a sample. The method comprises the steps of 1)combining a sample with a composition of lyotropic liquid or liquidcrystalline material; 2) binding said composition of lyotropic liquid orliquid crystalline material to a substrate; and 3) measuring for one ormore molecules of interest on said substrate. In some embodiments, themeasuring step is performed qualitatively. In other embodiments, themeasuring step is performed quantitatively. The step of binding may beperformed by said composition bonding directly to said substrate, e.g.by hydrogen bonding or ionic bonding. In some embodiments, sample is aliquid medium such as blood, serum, or urine. In one embodiment, thecomposition is combined with said sample in said combining step in theform of a plurality of particles. In another embodiment, at least two ofsaid plurality of particles include different capture molecules. Themeasuring step of the method may be performed in an assay such as, forexample, ELISA, MALDI or SELDI. In a preferred embodiment, the lyotropicliquid or liquid crystalline material is cubic phase. The molecule ofinterest may be a cancer marker.

The invention further provides a method of performing an assay, whichcomprises the steps of 1) combining a sample with a composition oflyotropic liquid or liquid crystalline material which has incorporatedtherein one or more capture molecules; 2) allowing one or more analytemolecules in said sample to bind with said one or more capture moleculesin said composition of lyotropic liquid or liquid crystalline material;3) binding said composition of lyotropic liquid or liquid crystallinematerial to a substrate; and 4) measuring the analyte molecules bound tosaid capture molecules. The step of measuring step may be performedqualitatively or quantitatively. In some embodiments, step of binding isperformed by said composition bonding directly to said substrate, e.g.via hydrogen bonding, or ionic bonding. In some embodiments, the sampleis a liquid medium such as, for example, blood, serum, or urine. In oneembodiment, the composition is combined with said sample in saidcombining step in the form of a plurality of particles. In yet anotherembodiment, at least two of said plurality of particles includedifferent capture molecules (for example, antigens and/or antibodies).Alternatively, the analyte molecules may be antigens or antibodies, andmay also be cancer markers. In some embodiments, the measuring step ofthe method performed in an assay such as ELISA, MALDI or SELDI. In apreferred embodiment, the lyotropic liquid or liquid crystallinematerial is cubic phase. The method may further comprise the step ofcoating the lyotropic liquid or liquid crystalline material with acoating, after the step of allowing said analyte molecules in saidsample to bind with said capture molecules and before the step ofbinding said composition to said substrate.

The invention further provides a composition used for calibration in anassay, the composition comprising a plurality of particles formed fromlyotropic liquid or liquid crystalline material, which bind directly toa surface of a substrate, each of said plurality of particles having atleast two different marker molecules present in the particle. In someembodiments, the particles are coated; in other embodiments, theparticles are uncoated.

The invention further provides a method for performing a laserdesorption ionization assay. The method comprises the steps of: 1)binding a lyotropic liquid or liquid crystalline material to a surfaceor substrate on which a sample is or will be deposited; 2) coating alayer of said lyotropic liquid or liquid crystalline material and saidsample with a chemical which crystallizes in situ to form an energyabsorbing matrix; and 3) measuring one or more compounds of interest insaid sample after said binding and coating steps using laser desorptionionization. In one embodiment, the step of coating is performed using achemical selected form the group consisting of cinnamic acid;cyano-4-hydroxy-cinnamic acid; 3,5-dimethoxy-4-hydroxycinnamic acid;hydroxycinnamic acid-3-phenylpropionic acid; caffeic acid; ferulic acid;2-(4-hydroxyphenylazo)-benzoic acid; 3-hydroxypicolinic acid; nicotinicacid; 2-pyrazinecarboxylic acid; 2,5-dihydroxybenzoic acid; succinicacid; sinapinic acid and its methyl and dimethyl esters and ethers;2-amino-4-method-5-nitropyridine; 2-amino-5-nitropyridine; and6-aza-2-thiothymine. In some embodiments, the binding is performed bysaid lyotropic or liquid crystalline material bonding directly to saidsubstrate, e.g. by hydrogen bonding or ionic bonding. The lyotropic orliquid crystalline material may incorporate therein one or more markermolecules. Alternatively, the lyotropic or liquid crystalline materialmay incorporate therein one or more capture molecules. In oneembodiments, the lyotropic or liquid crystalline material is bound tosaid substrate in the form of a plurality of particles. In oneembodiment, the particles are coated; in another, they are uncoated. Inone embodiment of the invention, the lyotropic liquid or liquidcrystalline material is combined with said sample prior to said steps ofbinding, coating and measuring. In a preferred embodiment, the lyotropicliquid or liquid crystalline material is cubic phase. In variousembodiments of the invention, the coefficient of variation in the methodis lowered by a factor of 5 or more in the presence of said lyotropicliquid or liquid crystalline material; or by a factor of 3 or more inthe presence of said lyotropic liquid or liquid crystalline material; orby a factor of 2 or more in the presence of said lyotropic liquid orliquid crystalline material. In some embodiments, the particle coatingcomprises a chemical from which said energy absorbing matrix is formed.In other embodiments, the particle coating comprises a chemical selectedform the group consisting of cinnamic acid; cyano-4-hydroxy-cinnamicacid; 3,5-dimethoxy-4-hydroxycinnamic acid; hydroxycinnamicacid-3-phenylpropionic acid; caffeic acid; ferulic acid;2-(4-hydroxyphenylazo)-benzoic acid; 3-hydroxypicolinic acid; nicotinicacid; 2-pyrazinecarboxylic acid; 2,5-dihydroxybenzoic acid; succinicacid; sinapinic acid and its methyl and dimethyl esters and ethers;2-amino-4-method-5-nitropyridine; 2-amino-5-nitropyridine; and6-aza-2-thiothymine. In one embodiment, the uncoated particles arecoated after combining with said sample but before said step of binding.

The invention further provides a method of preventing non-specificbinding during an assay that uses a solid support or substrate. Themethod comprises the steps of: 1) binding one or more capture moleculesto a surface of said support or substrate; and 2) binding lyotropicliquid or liquid crystal material to said surface of said support orsubstrate at locations on said surface where capture molecules are notbound, whereby samples exposed to said support or substrate arepresented with one or more regions for enabling specific binding saidone or more capture molecules and are blocked from non-specific bindingto said surface of said support or substrate by said lyotropic liquid orliquid crystal material. In one embodiment, the step of bindinglyotropic liquid or liquid crystal material is performed by depositingsaid lyotropic liquid or liquid crystal material over said surface ofsaid support or substrate after said step of binding one or more capturemolecules to said surface of said support or substrate. In anotherembodiment, the lyotropic or liquid crystalline material is bound tosaid support or substrate in the form of a plurality of particles. Insome embodiments, the particles are coated; in others, they areuncoated. In some embodiments, the step of binding said lyotropic orliquid crystalline material to said support or substrate is performed bybonding directly to said support or substrate (e.g. by hydrogen bondingor ionic bonding). In a preferred embodiment; the lyotropic liquid orliquid crystalline material is cubic phase.

The invention further provides a particle, comprising: 1) a lyotropicliquid or liquid crystalline matrix; 2) a first coating on a surface ofsaid lyotropic liquid or lyotropic liquid crystalline material; and 3) asecond coating on a surface of said first coating, said second coatingbeing different chemically and/or physically from said first coating.The second coating may be positively or negatively charged, and/or maybe capable of hydrogen bonding. A capture molecule and/or a molecularmarker may be incorporated in the particle. In a preferred embodiment,the lyotropic liquid or liquid crystalline material is cubic phase.

The invention further provides a particle, comprising: 1) a lyotropicliquid or liquid crystalline matrix; and 2) a constituent associatedwith said lyotropic liquid or liquid crystalline matrix which, uponactivation by a change in pH, temperature, or other physical or chemicalcondition, forms a coating on said lyotropic liquid or liquidcrystalline matrix, said coating causing said particle to bind directlyto a surface of a substrate. A capture molecule and/or a molecularmarker may be incorporated in the particle. In a preferred embodiment,the lyotropic liquid or liquid crystalline material is cubic phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A schematic representation of a marker molecule in materialbinding to a substrate used in a MALDI or SELDI type system.

FIGS. 2A and B. A schematic representation of capture molecules withbound analytes in material binding to A, an assay substrate, and B, aSELDI substrate.

FIG. 3. A schematic representation of material and sample together withmatrix material on a substrate used in a MALDI or SELDI type system.

FIG. 4. A schematic representation of material used as a blocking agenton an assay substrate.

FIG. 5. As discussed in Example 8, a plot of mass, spec intensities as afunction of the molecular mass to charge (m/z) ratio, forbiomacromolecules in a pool of serum from normal (cancer-free) humansubjects. Three preparations are plotted for each m/z. The left-most bar(solid) at each ratio is the case where serum was added to a simplebuffer, and the right-most bar (hollow) is the case where serum wasadded to a buffer-diluted (1:1000) dispersion of coated reversed cubicphase particles of material.

FIG. 6. This figure shows a schematic representation of the procedureand results obtained in the experiment described in Example 12.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Compositions of lyotropic materials (such as lyotropic liquids or liquidcrystalline materials), and methods for their use with respect tobinding to assay substrata are herein disclosed. In one embodiment,macromolecules such as captured markers or standards are containedwithin the lyotropic material, and the compositions stably bind tosurfaces such as those used for the substrata of many assay systems. Thestandards are thus immobilized on the assay surface by virtue of beingwithin the lyotropic material, and are protected from components ofbiological samples (e.g. proteases), and the accuracy of measurementsthat rely on the standards is thus enhanced. An exemplary assay surfaceis the surface of SELDI-MS chips. Exemplary standards includebiologically relevant macromolecular species such as proteins, peptides,dendrimers, nucleic acids, polysaccharides, etc.

Nanostructured lyotropic liquid and liquid crystalline phase materialssuitable for use in the present invention, have been described, forexample, in U.S. Pat. Nos. 6,482,517 and 6,638,621, (both to D MAnderson), the complete contents of which are hereby incorporated byreference. These patents describe coated particles of nanostructuredlyotropic liquid and liquid crystalline phase materials with preselectedsurface chemistries including ionic, hydrophobic, and hydrogen bonding,as well as the presence of antibodies, lectins, nucleic acids,receptors, chimera, avidin, and other biospecific targets at or near thesurface of the particles. Both materials and methods of making suchparticles are discussed in detail in these patents, which also describethe incorporation of macromolecules such as proteins in thenanostructured interior of the particles.

Such coated particles, with macromolecules of appropriate molecularweight(s) incorporated in the interior (and thus sequestered andprotected against potentially degrading influences such as proteases ornucleases), can further comprise a coating capable of binding to anassay substrate such as a SELDI substrate. Therefore, such coatedparticles are especially preferred in certain applications of thisinvention. In particular, ionically charged coatings such as ionicsurfactants or polyelectrolytes can be incorporated in the context ofthe instant invention, either as described in the methods of U.S. Pat.Nos. 6,482,517 and 6,638,621 for coating nanostructured liquid andliquid crystalline phase materials, or as second (or higher) coatingsupon first coatings achieved by those methods. For example, where U.S.Pat. No. 6,638,621 describes a method for producing a liquid crystallineparticle coated with a crystalline shell material ofzinc-acetyltryptophanate, the present disclosure describes a method forputting a second coating on such a coated particle (containing embeddedprotein markers), such as to achieve a strong, preselected ionic chargeor hydrophobically-interacting surface chemistry.

Thus, in this disclosure new compositions and methods are described inwhich coated particles are subjected to a second coating process, wherethe second coating is chosen, for example, for its plate-binding andlow-solubility characteristics. In general, it is much simpler to applya coating to a solid-coated particle than to an uncoated liquid orliquid crystalline particle. This yields particles with two,substantially nested, coatings, wherein the substantially outermostcoating binds effectively to the desired assay substrate. The first(inner) coating is selected on the basis of compatibility with thenanostructured liquid or liquid crystalline matrix, and may be formedusing the coating methodologies discussed in U.S. Pat. No. 6,638,621, oron the basis of pre-existing or to be discovered technology and practicefor making coated liquid or liquid crystalline particles. Once thiscoating has been applied, application of the second (outer) coating canproceed without limitations imposed by the liquid or semi-solid natureof the matrix, since this is now coated by a solid. Indeed, in thecourse of this work the application of a second coating was found to besurprisingly robust, particularly in the case where azinc-N-acetyltryptophan first coating was applied. A range of secondcoatings was applied under conditions that might have been incompatiblewith the particles absent the first zinc-NAT coating. Several of thesecoatings, and the particles so coated, were found to exhibit excellentbinding to SELDI plates of various surface chemistries.N-acetyltryptophan is known to have stabilizing effects on proteins (forexample, it is used to stabilize albumin, in several commerciallyavailable formulations of human albumin for injection), providinganother reason why it is a good choice for the first coating since thisis in direct contact with the nanostructured liquid crystalline (in thiscase) matrix containing the embedded protein.

Significantly, it has been discovered that certain compositions areable, in a turn-key fashion, to be used in the following protocolresulting in extremely high and sharp SELDI-MS intensities for acalibration peak:

1) contact the composition (in some cases after dilution), containing amacromolecule associated with a carrier particle, with a biologicallyrelevant material;

2) contact an appropriate substrate for an appropriate length of timewith the composition;

3) rinse the substrate.

This straightforward protocol results in a significantly greaterdeposition of the macromolecule on the substrate than occurs in theabsence of the carrier particle. In some embodiments of the invention,the macromolecule is greater than 1,000 in molecular weight, and is mostpreferably a peptide, protein, or less preferably a polysaccharide ordendrimer. The enhancement factor, namely the ratio of the peakintensity in the carrier particle-macromolecule system to that in theabsence of the carrier particle, is preferably greater than about 2,more preferably greater than about 10, and most preferably greater thanabout 100.

The following definitions and concepts will be useful.

“Assay”: in the context of the instant invention, an assay is aqualitative or quantitative measurement or detection of a specificsubstance of biological or biochemical importance. Furthermore, in thecontext of this invention the assays of interest are heterogeneous,since they involve a solid-phase substrate.

Examples of assay types for which the instant invention can be usefulare: assays based on mass spectrometry techniques, including but notlimited to MALDI-MS and SELDI-MS; ELISAs; radioimmunoassays;fluorescence immune assays; electrospray ionization mass spectrometry;chemiluminescent assays; surface plasmon resonance analysis; indirectimmunofluorescence assays; nucleic acid hybridization assays;polymerase-chain-reaction-based assays; multiplex assays; andchromatography-based assays. In the latter case, a chromatography beador bonded phase serves as the substrate to which particles of theinstant invention bind, and such an assay can be particularly useful inthat it can be preparative, allowing the extraction and/or purificationof a captured or encapsulated compound. Overall, the field of “biochips”is an exploding field that will continue to produce new assay techniquesand formats, many of which will be amenable to the materials and methodsof this invention.

“Substrate”: a substrate is a solid surface, not part of a livingorganism and thus substantially artificial, which has as its mainpurpose in the context of this invention to provide a controlled andwell-characterized surface for deposition of one or materials ofimportance in a diagnostic assay. While a substrate may contain one ormore biological components, its predominant solid or solid-like behavioris established by material that is far removed from living tissue, as,for example, paper is removed from the living tree from which is wasderived.

“Marker”: in the context of this invention, a marker is a compound whosepresence and level in an organism is (or in the case of an autopsy orarcheological investigation, can be) correlated with a particularphysiological condition, often though not always a disease or injurystate, or less commonly with drug usage, nutritional habits, stress, orother physiologic condition.

Peptides, proteins, and other compounds amenable to solid-phase assaysas discussed herein, which are of importance in current biomedicalpractice and research are well known in the art, and U.S. Pat. No.6,638,621 provides a listing of some of these compounds for whichantibodies are currently available. Especially preferred analytes are:TGF-alpha MMP-2, and IGF-II, thyrotropin (TSH), triiodothyronine,thyroxine, free thyroxine, follitropin, lutropin, prolactin, betasubunit of human chorionic gonadotropin, cortisol, ferritin,alpha-fetoprotein, carcinoembryonic antigen, and prostate-specificantigen, somatostatin, angiotensin, insulin, LHRH, CA125, TATI, andneuron-specific enolase (NSE). In addition to these well-characterizedmarkers, MALDI and SELDI analyses of serum from blood pools ofcancer-free and cancer patient groups has revealed key markers ofcertain cancer types that are not yet identified, and appear to befragments of proteins leftover from pathological lysis of proteins, andthese are of particular importance in the context of this invention.

“Lyotropic liquid crystalline phase”: lyotropic liquid crystallinephases include the normal hexagonal, normal bicontinuous cubic, normaldiscrete cubic, lamellar, reversed hexagonal, reversed bicontinuouscubic, and reversed discrete cubic liquid crystalline phases, togetherwith the less well-established normal and reversed intermediate liquidcrystalline phases. All of the lyotropic liquid crystalline phases arecharacterized by domain structures, composed of domains of at least afirst type and a second type (and in some cases three or even more typesof domains) having the following properties:

a) the chemical moieties in the first type domains are incompatible withthose in the second type domains (and in general, each pair of differentdomain types are mutually incompatible) such that they do not mix underthe given conditions but rather remain as separate domains;

b) the atomic ordering within each domain is liquid-like rather thansolid-like, lacking lattice-ordering of the atoms; (this would beevidenced by an absence of sharp Bragg peak reflections in wide-anglex-ray diffraction);

c) the smallest dimension (e.g., thickness in the case of layers,diameter in the case of cylinders or spheres) of substantially alldomains is in the range of nanometers (viz., from about 1 to about 100nm); and

d) the organization of the domains conforms to a lattice, which may beone-, two-, or three-dimensional, and which has a lattice parameter (orunit cell size) in the nanometer range (viz., from about 5 to about 200nm); the organization of domains thus conforms to one of the 230 spacegroups tabulated, for example, in the International Tables ofCrystallography, and would be evidenced in a well-designed small-anglex-ray scattering (SAXS) measurement by the presence of sharp Braggreflections with d-spacings of the lowest order reflections being in therange of 3-200 nm.

“Cubic phase”: Such a phase has cubic crystallographic symmetry, whichmakes it optically isotropic and yields characteristic indexings of theBragg peaks in SAXS, corresponding usually to one of the space groupsIm3m, Pn3m, or Ia3d. The bicontinuous property, in which both polar andapolar components are simultaneously continuous in all three dimensions,gives rise to high self-diffusion coefficients of all components of lowMW, whether they are segregated into the polar or the apolar domains,and also gives rise to high viscosities, often in the millions ofcentipoise. This phase generally appears at lower water contents thanlamellar phases, and/or at higher water contents than reversed hexagonalphases, and can also sometimes be induced by adding a hydrophobiccomponent to a lamellar phase, or a non-surfactant amphiphile with aweak polar group. When this is the phase used in the practice of thisinvention and it is desired to have this in contact with a solvent thenthe solvent should preferably be a polar one, typically water or aqueousbuffer, but more generally a polar solvent or mixture thereof. The poresize can be adjusted by changing the composition, and be determinedprecisely.

Some of the favorable features that distinguish reversed cubic phases asbeing especially preferred in the context of this invention are asfollows.

Lipid-dense, high internal surface area: with lipid concentrationstypically on the order of 30-50%, and every point in the cubic phaselying within a few nanometers of both an aqueous domain and a lipidbilayer, cubic phases are superior matrices for biomacromolecules. Itcannot be overstated that in contrast with the common misconception thata given molecule is situated, at any given moment, either in an aqueousor an oily domain, in actual fact most proteins have a strong propensityto situate so as to straddle the polar-apolar interface (the dividingsurface between the polar head groups and apolar chains of the lipid).NMR analyses have shown that even long-chain alcohols—highly hydrophobiccompounds with but a single polar group at their end—are situated sothat the hydroxyl group is strongly bound at the polar-apolar interface.In the case of biomacromolecules, a great deal is known about theregistry between polar and apolar epitopes of membrane-associatedproteins with those of the lipid bilayer. Specific surface areas intypical cubic phases, measured over the polar-apolar(hydrophilic-hydrophobic) interfacial surfaces, are in the range of 200m²/gm. As a result, typical loadings of proteins achievable in cubicphases are 30% by weight, and partition coefficients so high thatwater-phase concentrations are below detection limits. Together theseproperties of high loadings and high partition coefficients mean thatcrucial proteins including markers can be imbibed from assay solutionsand maintained in solution within the cubic phase. A protein can be saidto partition strongly into a liquid crystal when the partitioncoefficient, measured between the liquid crystal and aqueous buffer (asopposed to the traditional measurement between octanol and water) isgreater than about 100, more preferably greater than about 1,000 andmost preferably greater than about 10,000.

High bilayer fluidity: the bilayer fluidity (which refers specificallyto the microviscosity in the bilayer, and is substantially independentof the viscosity of the macroscopic material) of the cubic phase permitsdissolution and both orientational and diffusional freedom ofmacromolecules within the bilayer, and can be of critical importance inaffording the proper presentation of capture molecules. This is ofcrucial importance in the current invention.

Highly viscosity and pseudoplasticity: the three-dimensional,lattice-ordered supermolecular structure of cubic phases gives rise toextremely high zero-shear viscosities, measured in the billions ofcentipoise, but modest shear transiently breaks the structure andreduces the viscosity by many orders of magnitude. Cubic phases behaveessentially as solid-like materials under low-shear conditions, makingfor structural permanence that distinguishes them from thermally roiledmaterials such as micellar solutions and micelles, but that only mildshear conditions are needed to break the materials into microparticles.In the current invention, shear requirements can be further reduced byjudicious application of phase diagram information, circumventing theneed for high-pressure homogenization as required in, e.g., liposomeproduction. Delicate, shear-sensitive biopharmaceuticals can thereforebe encapsulated in robust, protective matrices without exposure to harshconditions.

Permselective accessible aqueous porosity: the nanoporous-networkstructure of the cubic phase, such that the 3-dimensional network ofaqueous pores lacing the entire particle is accessible from the outsidefor molecules smaller than the poresize, is a feature that clearlydistinguishes reversed cubic phase from liposomes and emulsions, byproviding ready accessibility of analytes and assay-associated moleculesto compounds in cubic phase particles and films. Due to the uniformporesize in these lattice-ordered materials, where said poresize can besubstantially pre-selected and tuned by composition over the size rangethat covers the range of protein dimensions, it is possible to formulateparticles that can allow the passage of peptides but exclude degradativeproteins such as proteases. This is of particular potential importancein the case of SELDI-based early cancer detection methodologies, wherethe markers have been found to be peptides and small fragments ofproteins.

Particle stability. Stability is one feature in which particles of theinstant invention excel over certain other materials, such as liposomesfor example. Uncoated particles of cubic phase, as exemplified by thepropofol dispersion described in Example 3 below, exhibit excellentlong-term (2+ years) stability when stored at room temperature, andexcellent accelerated stability (45° C.) over 9 months or more as well.Particle sizes as measured by light scattering show virtually no changeover the lifetimes cited. Particles coated with zinc-NAT, exemplified bya number of Examples below, exhibit long-term stability at roomtemperature, and furthermore stabilize sensitive actives by virtue ofthe coating. This stability is accomplished within the realm ofhigh-fluidity bilayer materials, as discussed above.

Particle shape. In contrast with liposomes, particles composed of cubicphase, whether coated or not, appear to have a strong tendency to assumepolyhedral forms, which can allow them to more intimately bind to solidsubstrata over a larger footprint. The polyhedral form is essentially amanifestation of a crystal habit, albeit in this case in the context ofa supermolecular liquid crystal, which nevertheless conforms to a cubiccrystallographic space group. This feature represents a distinctionfrom, and advantage over, spherical particles such as liposomes,particularly liposomes made from high-transition temperature lipids thatyield rigid bilayers, which do not conform well to surfaces in general.

Thus, milieu-sensitive proteins and biomacromolecules can be capturedand sequestered within dispersed cubic phase particles, protected byvirtue of permselectivity inherent in the accessible cubic phaseporosity and/or by one or more coatings, bound by capture molecules thatare virtually assured of near-physiologic conformation, and deposited ona substrate through particle-substrate affinity that can be independentof capricious protein-substrate interactions. It should be noted thatthe ability of a particle to deposit a particular compound onto asubstrate independently of the compound-substrate interaction (that is,independently of whether or not it would bind without a particle beingpresent) is at least to some extent dependent on having a verylow-solubility coating. In view of the high dilutions that are typicallyused in ultrasensitive techniques like SELDI, even a small aqueoussolubility of the particle coating can result in the stripping away ofthe coating and contact between the compound and substrate, introducingcompound-substrate interactions back into the picture.

“Bilayer-associated”, “membrane-associated”: A compound or moiety isbilayer-associated if it partitions preferentially into a bilayer overan aqueous compartment. Thus, if a bilayer-rich material such as areversed cubic phase material exists in equilibrium with excess waterand is placed in contact with excess water, and a bilayer-associatedcompound or moiety is allowed to equilibrate between the two phases,then the overwhelming majority of the compound or moiety will be locatedin the bilayer-rich phase. The concentration of the compound or moietyin the bilayer-rich phase will be at least about 100 times, andpreferably at least about 1,000 times, larger than in the water phase.

It is important to note that although the reversed hexagonal phases andreversed discrete or discontinuous cubic phases do not have a truebilayer as the fundamental structural unit, in the present disclosure wewill nevertheless use the term “bilayer-associated” to describecomponents that partition into the lipid-rich (or surfactant-rich)microdomains irrespective of whether such domains are considered“monolayers” or “bilayers”. The term “bilayer-associated” is thus moredirected to the partitioning of the compound in question than to theprecise nature of the lipid (or surfactant) region.

Besides capture and bilayer-charging compounds, another component of theparticle that can be bilayer-associated is the biomolecule or standarditself. For small molecules, this is preferred, since it means that thebiomolecule will tend to remain with the particle even when the particleis exposed to large volumes of biological fluids. However, biomoleculesthat partition preferentially into the aqueous channels of the reversedliquid crystalline material, including many if not most proteins andother biomacromolecules, can be incorporated into particles utilized inthe current invention, as can biomolecule that localize to comparableconcentrations in the aqueous and hydrophobic compartments. Indeed, oneimportant aspect of the invention which distinguishes it over typicalemulsions, for example, is the very large polar-apolar surface areas,which provide ample volume for biomolecules which have apolar groups orepitopes that prefer a hydrophobic milieu as well as polar groups thatprefer the hydrophilic milieu of the aqueous channels and headgroup-rich regions.

“Energy-absorbing matrix”: For MALDI-MS, SELDI-MS, and relatedapplications, “energy-absorbing matrices” are those that can serve asthe matrix which interacts with laser light to break up the material onthe substrate and fly (propel) the material down the flight tube.Examples of such coatings include the following acids: cinnamic acid;cyano-4-hydroxy-cinnamic acid; 3,5-dimethoxy-4-hydroxycinnamic acid;hydroxycinnamic acid-3-phenylpropionic acid; caffeic acid; ferulic acid;2-(4-hydroxyphenylazo)-benzoic acid; 3-hydroxypicolinic acid; nicotinicacid; 2-pyrazinecarboxylic acid; 2,5-dihydroxybenzoic acid; succinicacid; and sinapinic acid and its methyl and dimethyl esters and ethers.Bases are also used, such as 2-amino-4-methyl-5-nitropyridine and2-amino-5-nitropyridine; 6-aza-2-thiothymine.

Methods and Materials.

An important aspect of the instant invention is the crafting oflyotropic liquid or, more preferably, liquid crystalline material, so asto bind to a substrate either as a collection of particles or as a film,which in turn may or may not be coated. In some cases, the lyotropicmaterial and the substrate will be chosen together, in tandem, so as toyield the desired binding. This can be accomplished by judicious use ofone or more of the following three general approaches:

-   -   A) coating: particles are at least partially covered with a        coating material that is selected so as to bind to the        substrate;    -   B) compound in the lyotropic material: the lyotropic material is        chosen so as to incorporate one or more compounds that promote        binding of the material to the substrate; most preferably these        compounds are bilayer-associated; less preferably, a        non-bilayer-associated compound in the lyotropic material is        retained in the material by a gelation step that is carried out        within the lyotropic material;    -   C) hydrophobic interaction: the lyotropic material and substrate        are selected in such a way that a hydrophobic interaction        between the two promotes binding.        Detailed descriptions of methods for producing particles of        lyotropic materials coated with a wide range of coating        materials are given in U.S. Pat. No. 6,482,517 and U.S. Pat. No.        6,638,621, the contents of which are incorporated herein by way        of reference, and the discussion below describes how such        methods can be incorporated into embodiments of the instant        invention. Similarly, concerning the second approach, detailed        descriptions of methods for producing uncoated particles of        lyotropic materials containing bilayer-associated compounds, as        well as capture compounds such as antibodies and lectins, are        given in U.S. application Ser. Nos. 10/889,313 and 10/170,214.        Whereas the latter reference teaches the use of particles        incorporating capture (or “target”, in the terminology used in        that disclosure) molecules in particle interiors for the purpose        of capturing molecules in solution for liquid-phase assays, the        instant invention by contrast teaches, among other things, the        incorporation of capture molecules at particle surfaces in order        to obtain binding of particles to properly selected solid        substrata for solid-phase assays. Concerning methods of gelation        inside lyotropic materials, U.S. Pat. No. 5,244,799 and,        particularly, U.S. Pat. No. 5,238,613, the contents of which are        hereby incorporated by reference, describe methods and materials        for accomplishing this, and this is discussed in the context of        the instant invention below.

This approach first involves the selection of a pair of materials, oneto serve as the substrate and the other as a coating on a coatedlyotropic liquid or liquid crystalline material, or as a particle-boundcompound at the surface of (and possibly inside of, as well) an uncoatedparticle, or as the lipid or surfactant in an uncoated particle designedfor hydrophobic interaction-based binding. In many cases, the choice ofsubstrate will be in part, or fully, driven by considerations that havenothing to do with the lyotropic material. For example, one may bedealing with a substrate that is chosen so as to bind certain compoundsindependently of (and perhaps along with) any lyotropic particles ormaterials, or to be compatible with certain experimental conditions suchas high local radiation intensities, low nonspecific bindingcharacteristics, or the like. In any case, with a given substrate inmind, the invention contemplates the judicious choice and application ofparticle composition, in particular of the particle surface, so as toachieve the desired particle deposition and binding.

The substrate-particle binding in the instant invention will be achievedby virtue of one or more favorable interactions selected from the groupthat includes the following: electrostatic (anion/cation pairing),hydrophobic interaction, hydrogen bonding, polymer bridging, surfacedehydration, van der Waals attraction involving a high-energy surface,magnetic, antibody/antigen, lectin/saccharide, nucleicacid/complementary nucleic acid, receptor/ligand, and other proteinbinding interactions such as avidin/biotin, etc. (Here the forward slashterminology A/B means A binding to B). The preferred binding mechanisms,both in the case of coated and uncoated particles, for typicalapplications, will be those involving strong, non-biospecific attractiveforces such as electrostatic and hydrophobic interaction forces, becausebiospecific mechanisms such as receptor/ligand interactions typicallyinvolve more delicate proteins that can denature, e.g., when adsorbed toa solid-coated particle, absent more sophisticated tethering schemes.However, in more critical applications, particularly those that justifyexpensive and/or elaborate chemistries (e.g., protein PEGylation orchimeras), biospecific mechanisms can become the preferred means, withantibody/antigen interactions being most preferred.

Generally, in the case where coated particles are used in the currentinvention with the intention that they bind to the substrate as coatedparticles (that is, where the coating is the binding entity), thecoating material should have a solubility in water of less than about5%, more preferably less than about 1%, and most preferably less than,about 0.1%, so that the coating does not substantially dissolve when theparticles are applied in the assay, which will nearly always beaqueous-based. In case the solubility is not sufficiently low to avoidunwanted dissolution, then the water used in the assay system must besaturated in the coating compound, though this is far less preferred dueto deleterious salting-out and dissolution-recrystallization effects.Contrariwise, if the intent is that the particles be formulated andstored as coated particles (e.g., for purposes of stabilization via thecoating) but that the coating should dissolve so as to leave uncoatedparticles for the application, then the reverse is true, and the coatingsolubility should be greater than about 0.01%, and more preferablygreater than about 0.1%. In the case of uncoated particles, the crucialcomponents of the lipid (or surfactant) bilayer should have a watersolubility less than about 5%, more preferably less than about 1%, andmost preferably less than about 0.1%.

Various particle types and means of binding are now discussed.

Electrostatic Binding.

In SELDI applications, as well as in a wide range of other analyticaltechniques, it is very common to utilize substrata whose mostcharacteristic feature is a significant surface charge. Indeed, at thecurrent stage of SELDI at the time of this writing, this is by far themost prevalent case. Typically the preferred substrate surface charge iscationic (otherwise known as anion-exchange), since most biologicalcomponents are negatively-charged at pH values near physiological.Anionic (i.e., cation-exchange) substrata are selective towards the farless common cationic compounds.

In this approach, the surface of the lyotropic particle will beengineered so as to be oppositely charged as the substrate, and zetapotential measurements can be used to determine the chargeexperimentally. To avoid undue experimentation, this disclosuredescribes methods and materials for engineering such a particle.

Compounds containing the following chemical groups can be charged,albeit weakly and/or over a fairly narrow pH range: silanol, aldehyde,ketone, carboxylic ester, carboxylic acid, isocyanate, amide, acylcyanoguanidine, acyl guanylurea, acyl biuret, dimethylamide,nitrosoalkane, nitroalkane, nitrate ester, nitrite ester, nitrone,nitrosamine, pyridine N-oxide, nitrile, isonitrile, amine borane, aminehaloborane, sulfone, phosphine sulfide, arsine sulfide, sulfonamide,sulfonamide methylimine, alcohol (monofunctional), ester(monofunctional), secondary amine, tertiary amine, mercaptan, thioether,primary phosphine, secondary phosphine, and tertiary phosphine. Weaklycharged coatings are preferred in the present invention in the followingcontexts: to achieve electrostatic binding while minimizingelectrostatic repulsions, and/or unwanted electrostatic attractions,between particles and analytes; as first coatings in doubly-coatedparticle systems; and for binding the combines electrostatic withhydrophobic-interaction attractions.

Compounds containing the following chemical groups can be stronglycharged over some pH range (typically large), are:

-   -   a. Anionic groups: carboxylate (soap), sulfate, sulfonate,        sulfonate, thiosulfate, sulfinate, phosphate, phosphonate,        phosphinate, nitroamide, tris(alkylsulfonyl)methide, xanthate;    -   b. Cationic groups: ammonium, pyridinium, phosphonium,        sulfonium, and sulfoxonium.        The use of coatings that incorporate these chemical groups can        provide electrostatic stabilization of the particles in        dispersed form as during storage and in capture processes prior        to substrate binding, as well as electrostatic binding to the        substrate of opposite charge. Zeta potential measurements (e.g.,        employing laser Doppler electrokinetic measurements) are        important for validating and quantifying the charge on such        particles, and preferably conditions in the exterior aqueous        phase are chosen in such an experiment to match reasonably        closely the conditions that will be present in the actual        binding event, in the application.

Doubly-Coated Particles.

As discussed above, an especially effective method for producingparticles of this invention is to use an approach in which coatedparticles are subjected to a second coating process, where the secondcoating is chosen for its plate-binding and low-solubilitycharacteristics; as discussed above, it is much simpler to apply acoating to a solid-coated particle than to an uncoated liquid or liquidcrystalline particle. This second coating can be applied to coatedparticles using a wide range of means, including chemical precipitation,spray-drying (e.g., spray-drying a dispersion of coated particles withsecond coating dissolved or dispersed therein), spray-congealing,fluidized bed coating, electrospinning, sputter-coating,ion-bombardment, etc. Generally, nearly all of the methods discussed inU.S. Pat. No. 6,638,621 for putting a first coating on a lyotropicmaterial after it has been dispersed, can be also applied to coatedparticles for putting on a second coating. Chemical precipitation is themost preferred, with precipitation accomplished conveniently by a simpleacid-based reaction or counterion exchange. In many cases, the secondcoating can even be the same charge (cationic, anionic) as the firstcoating, and this is preferred if bridging and flocculation isexperienced when an oppositely-charged second coating is used. In thecase where a charged second coating is desired, most preferably thefirst coating is weakly- or un-charged (as is the case with theweakly-charged zinc-NAT first coating used in many of the Examplesbelow).

Especially preferred second coating materials are polymers, lipids, andsurfactants of low solubility, including divalent-ion salts andprotonated forms of anionic surfactants. Suitable lipids includephospholipids (such as phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, or sphingomyelin), or glycolipids (such asMGDG, diacylglucopyranosyl glycerols, and Lipid A.) Other suitablelipids are phospholipids (including phosphatidylcholines,phosphatidylinositols, phosphatidylglycerols, phosphatidic acids,phosphatidylserines, phosphatidylethanolamines, etc.), sphingolipids(including sphingomyelins), glycolipids (such as galactolipids such asMGDG and DGDG, diacylglucopyranosyl glycerols, and Lipid A), cholicacids and related acids such as deoxycholic acid, glycocholic acid,taurocholic acid, etc., and low-solubility salts thereof, gentiobiosyls,isoprenoids, ceramides, plasmologens, cerebrosides (includingsulphatides), gangliosides, cyclopentatriol lipids, dimethylaminopropanelipids, preferably double-chained or with saturated long chains (14 ormore carbons, preferably 16 or more). Preferred surfactants for secondcoatings are:

anionic—divalent salts and acid forms of alkyl sulfates, dialkylsulfosuccinates, alkyl lactylates, and carboxylate soaps of the formIC_(n) where either: a) the chain is saturated and the length n isbetween 14 and 20 and I is a monovalent counterion such as lithium,sodium, potassium, rubidium, etc.; or b) the chain is unsaturated orbranched, or of length n less than about 14, and I is a multivalentcounterion;

cationic—dimethylammonium and trimethylammonium surfactants withchloride, bromide or sulfate counterion, myristyl-gamma-picoliniumchloride and relatives, where single-tailed quaternary ammoniumsurfactants have saturated chains with lengths between about 14 and 20carbons, and double-tailed quaternary ammonium surfactants havesaturated chains with lengths between about 10 and 20 carbons;

Polymers preferred for the second coating are: polypropylene oxide,polybutadiene, polyisoprene, polyacrylic acid and its salts,polymethacrylic acid and its salts, polymethylmethacrylatepolyacrylamide, polyisopropylacrylamide, polyacrylonitrile, polyvinylacetate, polyvinyl caprylate, polystyrene, polystyrene sulfonic acid andits salts, pectin, chitin, chitosan, cellulose derivatives, alginic acidand its salts, gum arabic and its salts, gelatin, PVP, tragacanth, agar,agarose, guar gum, carboxymethylcellulose, arabinogalactan, Carbopol,chitin, chitosan, Eudragits, glycogen, heparin, pectin, and complexcarbohydrates which can, e.g., bind with specificity to varioussaccharide-recognizing compounds such as lectins. Chitosan and certainamino-containing Eudragits are of particular importance because they areamong the relatively small list of conveniently-available polymers whichare cationic, making them useful for binding to anionic substrata.

Uncoated Charged Particles.

For the purpose of binding uncoated particles to cationic (ion-exchange)substrata, a very effective approach is to incorporate into thelyotropic liquid or liquid crystalline material a bilayer-associatedanionic compound (and associated counterion), which is sufficient toestablish a significantly negative zeta potential on the particles. Inview of the high dilutions that are frequently employed in assays, thepartition coefficient of this compound in the particle over water needsto be very high, greater than about 100, more preferably greater thanabout 1,000 and most preferably greater than about 10,000. Fortunatelythis is the case for a wide range of charged compounds, both anionic andcationic, due to the high interfacial area property of these materials,especially cubic phases, as discussed above.

Especially preferred anionic moieties are: docusate, dodecylsulfate,deoxycholic acid (and related chocolates, such as glycocholate),tocopherol succinate, stearic acid and other 18-carbon fatty acidsincluding oleic, linoleic, and linolenic acids, gentisic acid,hydrophobic amino acids including tryptophan, tyrosine, leucine,isoleucine, aspartic acid, cystine, and their N-methylated derivatives,particularly N-acetyltryptophan, myristyl gamma-picolinium chloride,phosphatidylserine, phosphatidylinositol, phosphatidylglycerol(particularly dimyristoyl phosphatidylglycerol), and other anionic andacidic phospholipids. The person with skill in the art will recognizedocusate as the anionic moiety of the surfactant docusate sodium (alsoknown as Aerosol OT), and dodecylsulfate as the anionic moiety of thesurfactant sodium dodecylsulfate, or SDS. Surface-active polypeptidesand proteins, such as casein and albumin, may also be used, althoughcareful attention must be paid to the pH, which will have an effect onthe charge of the molecule.

Other compounds that can provide the anion include ascorbyl palmitate,stearoyl lactylate, glycyrrhizin, monoglyceride citrate, stearylcitrate, sodium stearyl fumarate, JBR-99 rhamnolipid (and otherbiosurfactants from Jeneil Biosurfactant), glycocholic acid, taurocholicacid, and taurochenodeoxycholic acid.

Other preferred anionic surfactants are: sodium oleate, sodium dodecylsulfate, sodium diethylhexyl sulfosuccinate, sodium dimethylhexylsulfosuccinate, sodium di-2-ethylacetate, sodium 2-ethylhexyl sulfate,sodium undecane-3-sulfate, sodium ethylphenylundecanoate, carboxylatesoaps of the form IC_(n), where the chain length n is between 8 and 20and I is a monovalent counterion such as sodium, potassium, ammonium,etc.

Cationic Bilayer-Associated Compounds.

For binding to anionic substrata, cationic bilayer-associated compoundsfor incorporation into uncoated lyotropic particles include:myristyl-gamma-picolinium chloride, benzalkonium chloride, tocopheryldimethylaminoacetate hydrochloride, Cytofectin gs,1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane, cholesterol linkedto lysinamide or ornithinamide, dimethyldioctadecyl ammonium bromide,1,2-dioleoyl-sn-3-ethylphosphocholine and other double-chained lipidswith a cationic charge carried by a phosphorus or arsenic atom,trimethyl aminoethane carbamoyl cholesterol iodide,O,O′-ditetradecanoyl-N-(alpha-trimethyl ammonioacetyl) diethanolaminechloride (DC-6-14),N-[(1-(2,3-dioleyloxy)propyl)]-N—N—N-trimethylammonium chloride,N-methyl-4-(dioleyl)methylpyridinium chloride (“saint-2”), lipidicglycosides with amino alkyl pendent groups,1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide,bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide,N-hexadecyl-N-10-[O-(4-acetoxy)-phenylundecanoate]ethyl-dimethylammoniumbromide, 3-beta-[N—(N′,N′-dimethylaminoethane)-carbamoyl, andparticularly useful is didodecyldimethylammonium bromide.

Other Useful Bilayer-Associated Compounds.

Other suitable charged bilayer-associated compounds for use in uncoatedparticles of the instant invention, which can take up a charge under atleast some conditions, include: fatty acids, phenolic compounds such aseugenol, isoeugenol, quinolines, hydroxyquinolines and benzoquinolines,tricyclics such as carbazole, phenothiazine, etc., pigments,chlorophyll, certain natural oil extracts particularly those which arephenolic (such as clove oil, ginger oil, basil oil), biosurfactants(such as Jeneil's “JBR-99”), a wide range of dyes. Amphiphilic proteinsand polypeptides can be used, including gramicidin, casein, albumin,glycoproteins, lipid-anchored proteins, receptor proteins and othermembrane proteins such as proteinase A, amyloglucosidase, enkephalinase,dipeptidyl peptidase IV, gamma-glutamyl transferase, galactosidase,neuraminidase, alpha-mannosidase, cholinesterase, arylamidase,surfactin, ferrochelatase, spiralin, penicillin-binding proteins,microsomal glycotransferases, kinases, bacterial outer membraneproteins, and histocompatibility antigens. As is well known, everyprotein has a net charge except at its isoelectric point (pI), and thusa membrane-associated protein is suitable for use in the presentinvention as long as the pH is away from its isoelectric point. A fewsuch proteins are currently accepted as inactive ingredients forpharmaceutical preparations, at least under some conditions, and theseinclude gluten, casein, and albumin.

Charged Gels.

As introduced above, another method for establishing a retained ioniccharge on an uncoated lyotropic particle, which need not involvebilayer-associated compounds, is to perform a gelation of a chargedcompound (monomer, oligomer, prepolymer, gelling polymer) inside thepores of a lyotropic material. Nature provides a number of chargedpolymers (typically polysaccharides) which can be gelled under mildconditions. These include gelatin, guar gum, pectin, alginic acid andits salts, gum arabic and its salts, tragacanth, agar, agarose,glycogen, heparin and the semisynthetic compoundscarboxymethylcellulose, Carbopol, chitosan, Eudragits, as well as anumber of proteins which can be made to gel, such as casein, gluten, andalbumin (note that the latter tend to be membrane-interactive by virtueof amphiphilicity). The gelation can be carried out in the bulklyotropic material, and the resulting gelled material then dispersed.Alternatively, if the dispersing can be performed before the gelationstep without leading to gelation outside the particles, then that ispreferred.

Hydrophobic Interaction-Based Binding.

Hydrophobic interaction is an excellent choice in general for the use ofthe instant invention in SELDI and other assays, since the particles ofthe invention are inherently well suited for this mechanism of binding.Since lyotropic liquids and especially liquid crystals are rich insurfactant, and in fact depend on hydrophobic interactions between thecomponents for their structure, they bind well to hydrophobicinteraction substrata. Furthermore, coating materials are, virtually bydefinition in these embodiments, of low-solubility in water, in order toperform their job as coatings. Thus, whether coated or uncoated, thelyotropic materials discussed in this disclosure are quite broadlywell-suited for HI-based binding to a substrate. This is demonstrated inExample 2 below where the “H50-8” SELDI chips showed a modest butsignificant amount of binding with two of the embodiments of thisinvention. Doubly-coated particles in which the second (outermost)coating is hydrophobic can be especially effective at binding, and canbe stabilized in aqueous dispersion by the adsorption of very smallamounts of surfactant, retaining the HI-based binding of the particles.

Biospecific Binding.

The methods discussed herein, as well as those in U.S. Pat. No.6,638,621 describing coated particles incorporating proteins and otherbiomolecules, and U.S. application Ser. No. 10/889,313 describinguncoated particles likewise incorporating biomolecules, can be used aspart of a methodology in which one member of an A/B biospecific bindingpair (e.g., A=antibody, B=corresponding antigen) is incorporated intothe particles, and the other immobilized on the substrate. “Biochips”incorporating attached biomolecules of importance in assays are becomingincreasingly used in a number of fields, so that a range of biomoleculesubstrata are becoming available. Selecting such a substrate and apaired compound for biospecific binding to the biomolecule bound on thatsubstrate, one is then faced with the job of producing a lyotropicparticle, coated or uncoated, incorporating that paired compound. Themethods of U.S. Pat. No. 6,638,621 and Ser. No. 10/889,313 can beapplied to solve that problem, and in the context of the presentinvention this is simplified relative to the cases of focus in thosedisclosures, because there is little if any need to limit oneself toformulations containing only low-toxicity compounds, as was required inthe pharmaceutical applications of most focus in those disclosures.

Other Compound Pairs.

In addition to the above means of establishing binding, the inventioncan take advantage of certain specific compound pairs that bindtogether, in spite of the fact that they may not neatly fit into one ofthe paradigms discussed above. For example, vitamin B12 (cyanocobalamin)is known to bind tightly to talc and other silicates. Another usefulapproach is to use a coating that is the same material as, or similarto, the substrate material, and to adsorb, to the surface of thecoating, a compound that binds both to the coating and to the substrate.For example, vitamin B12 binds to silicates. Therefore, if the substrateis a silicate, then a silicate-coated particle could be created, towhich B12 would be adsorbed, and the particle would be expected to bindto a silicate substrate, with the B12 acting as a sort of “glue” betweenthe two silicate surfaces.

Magnetism-Based Binding.

There are several potential means of producing particles with metalliccomponents that could be directed with magnetic fields to collect atsubstrate surfaces:

-   -   1) Metal coatings on lyotropic materials can be produced by        electrodeposition methods, or with the use of reducing agents.    -   2) Electroplating can deposit metal not only at the surface of,        but even in the interior pores of, porous lyotropic materials.    -   3) Metal particles, particularly nanoparticles, can be embedded        into lyotropic materials and particles by vigorous mixing.    -   4) Metal nanoparticles could be adsorbed onto the surface of        lyotropic particles, in analogy with Pickering emulsions.

Binding to Gold Substrata.

Gold is a particularly important substrate surface material. Itsinertness, well-characterized nature, high surface energy, and abilityto bind albumin are important motivations for using this substrate. Whencitrate ions are present, albumin can bind to gold surfaces viaelectrostatic interaction, but albumin binding to gold can occur even inthe absence of citrate. Citrate-coated gold is also used as a substratein some applications. Gold surfaces are often anionic, and the methodsand materials described herein for binding electrostatically to anionicsurfaces then apply. Thiol compounds can also be attached to goldsurfaces, including thioesters and thiocarbonates. In the presentinvention, promotion of particle binding to gold surfaces can beaccomplished by the incorporation of thiol-containing compounds, underconditions that promote attachment of thiol compounds to gold, which arewell known; the thiol-containing compound can be incorporated intolyotropic particles in one of three basic ways:

-   -   1) by incorporating into the lyotropic material a        bilayer-associated thiol compound, which exhibits a high degree        of partitioning into the lyotropic material; a thiol-containing        lipid, or hydrophobic or amphiphilic protein (with one or more        cysteine residues), can provide the required group, preferably        on an uncoated particle.    -   2) by incorporating a thiol-containing compound in the solid or        polymeric shell of a coated (or doubly-coated) particle; or    -   3) by incorporating a thiol group on a tether that is attached,        adsorbed, or partially imbibed within, a particle of this        invention.

Particles Coatable In Situ.

Particles that can be converted to coated particles by a chemicalreaction, for example upon change in, pH and/or addition of a divalention such as Zn²⁺, can be of particular advantage in this invention. Forexample, a particle of cubic phase containing embedded capturemolecules, such as antibodies or receptors, can allow access to aparticular compound (e.g., antigen or ligand, resp.) before the chemicalchange which induces the coating, and then after the chemical change andconversion to a coated particle, the advantages of coated particles aregained: permanence, protection of contents, desired bindingcharacteristics, etc. This is demonstrated in Example 12 below, whereuncoated particles imbibe fluorescently-labelled albumin and are thencoated by simply adding a zinc salt. Coated particles, and ways ofmaking them, are reported in U.S. Pat. No. 6,638,621, and these canyield uncoated-coated convertible particles in the following way. Acomposition and method of making a coated particle are chosen from thematerials and methods given in U.S. Pat. No. 6,638,621, with onecriterion being that the method involves first creating a dispersion ofuncoated particles, and then forming the coating on the dispersedparticles. This coat-forming step inevitably involves some change incondition, such as pH, temperature, pressure, salinity, divalent ion,addition of a reactant, removal of a solvent, etc. This coat-formingcondition change is then invoked subsequent to the imbibition or othercapture of the desired compounds by the uncoated particles, inducing thecoating of the particle preferably with captured compound inside. Insome applications it will be important to choose a coat-forming triggerthat will not cause denaturation or other diminishment of themolecule(s) of interest. As an example, in the case of the zinc-NATcoatings described in Example 1, the formation of the coating involvesthree conditions, any one of which can be used as the trigger: additionof NAT, addition of a zinc salt (such as zinc acetate), and addition ofbase. In this instance, the latter two are preferred because theaddition of NAT (as a soluble salt, e.g., with diethanolamine or NaOH)to the cubic phase in water not only aids in the dispersing of the cubicphase into particles, but also preferentially pre-localizes the NAT ator near the surface of the particles which then results in efficientcoating upon conversion to zinc-NAT.

Albumin-Bound Markers.

In recent years, SELDI- and MALDI-based research on markers of cancerand other diseases have shown that peptides and proteins of importanceas markers often bind to albumin in the blood. This appears to be such apredominant phenomenon that the choice of substrate is often drivenlargely by the requirement that it bind albumin optimally. In view ofthis, particles of the instant invention, most preferably uncoated cubicphase particles, which are able to adsorb or, more preferably, to imbibealbumin are potentially of particular importance. Example 12 belowprovides one case where albumin is readily imbibed into uncoated cubicphase particles, and furthermore that Example teaches how such particlescan be subsequently coated by the simple addition of a salt, thusencapsulating the albumin. One skilled in the art will recognize thatthe conditions used in Example 12 are mild enough that one would expectmany, if not most, of the albumin-bound material in the current contextto also be encapsulated, in such a process. Discussed herein is also thefact that certain substrata do bind the coated particles created inExample 12.

Another approach possible in the context of this invention is toincorporate into the particles one or more compounds capable of bindingalbumin, and capturing albumin-associated peptides and materials aswell. For example, a mouse anti-human antibody to albumin could beincorporated into, preferably, an uncoated particle.

Interactions of Lyotropic Particles with Energy-Absorbing Matrices.

As seen herein, the instant invention can greatly improvereproducibility and uniformity in MALDI and SELDI applications,accomplishing a reduction in the coefficient of variation (CV) by factorof 2, more preferably by a factor 3 or more, and most preferably by afactor of about 5 or more. The improvement—in some cases dramatic, asseen in Example 8 below)—in reproducibility (and peak intensities aswell) due to the application of the current invention may be due to oneor more of the following benefits that the invention can provide:

-   -   A) In a traditional SELDI assay, the adsorption of proteins to        the plate is a dynamic, first-come-first-serve competitive        process, which would be expected to yield diminished        reproducibility, high CV, whereas in contrast, the imbibition of        proteins into a lyotropic particle very quickly reaches        equilibrium, due to the fact that the diffusion distance to (and        into the interior of) the nearest particle is one micron, rather        than several hundred microns as with the normal SELDI assay, by        first principles, an equilibrium (or near-equilibrium) process        will generally lead to higher reproducibility, lower CV. A        several-fold reduction in CV can also be accomplished even if        the analyte molecules are adsorbing to the particle surface        rather than imbibing into the interior, although the more        dramatic improvements would be expected in the latter cases.    -   B) The presence of lyotropic liquid crystalline particles,        preferably those of very high low-shear viscosities, could be        improving the nucleation and growth characteristics, and        ultimate crystal size and uniformity of deposition, of the        energy-absorbing matrix material. Evidence for this is the fact        that the crystallization of zinc-NAT (as well as other coatings)        is obviously profoundly affect by the presence of dispersed        cubic phase particles in our production process for zinc-NAT        coated particles—specifically, the crystals take the form of an        ultrafine coating on the particles, rather than large,        supermicron crystals as is seen in a simple crystallization of        zinc-NAT. Indeed, Example 13 below reports how one        energy-absorbing matrix material is similarly crystallized in        the form of an ultrafine particle coating. If the nucleation,        growth, diffusion, or adsorption characteristics of        energy-absorbing matrix material is affected in such ways, and        particularly if this results in a more intimate association and        thus energy transfer between the energy-absorbing matrix and the        analyte molecules (which may in turn have imbibed into the        particles), then any of these effects could improve peak        intensities and reproducibilities. FIG. 3 is intended to depict,        schematically, the situation in which the energy-absorbing        matrix is intermingled with lyotropic materials of this        invention.    -   C) Since several of the Examples demonstrate that the current        invention strongly inhibits non-specific binding, the deposition        of proteins and peptides onto the substrate prior to coating is        expected to be more predictable and reproducible in the presence        of the particles of this invention.    -   D) The same inhibition of NSB can give rise to more efficient        desorption from the substrate (the “D” in MALDI and SELDI).

Lyotropic Particles Coated with Energy-Absorbing Matrices.

Another important type of embodiment of the instant invention involvesthe use of coated particles, in which the coating actually comprises anenergy-absorbing matrix material. Example 13 in fact demonstrates such aparticle. The particles can either be supplied in coated form, or can becoated in situ, atop the substrate, after imbibition of analyte inparticular (as demonstrated for the case of zinc-NAT coatings in Example12). Such materials and methods could offer one or more of the followingadvantages over the prior art practice of MALDI and SELDI:

-   -   A) The need for applying the matrix would be circumvented,        simplifying and shortening the procedure.    -   B) Also circumvented would be the organic solvents        (acetonitrile, etc) typically used to deposit energy-absorbing        matrices in the prior art, and which can result in loss of        information due to, e.g., solvent-mediated breakup of intimate        protein/peptide complexes.    -   C) In the case where analyte is sequestered inside of particles        subsequently coated, the intimate association between        energy-absorbing matrix and analyte can result in superior        energy transfer to the analyte and thus more efficient        desorption and detection.

More on Uncoated Particles.

Clearly, uncoated particles are of considerable utility in thisinvention, particularly uncoated particles with significantelectrostatic charge. Particles of reversed liquid crystalline phasematerial, particularly reversed cubic phase and to a lesser extentreversed hexagonal phase, can be stabilized in dispersion by a zetapotential which is greater in magnitude than about 25 millivolts, ormore preferably greater than about 30 mV, and this same charge can, inthe instant invention, induce binding of the particle to theappropriately chosen SELDI chip. The electrostatic charge is preferablyinduced by the incorporation, in the liquid crystalline material, of anionic bilayer-associated compound. Ionic surfactants, and chargedcompounds with relatively high octanol-water partition coefficients, areincorporated into the liquid crystalline material in order to establishthe charge. For the cases where an ionic additive is needed (that is,where the liquid crystalline phase itself does not have a chargedsurfactant as its main surfactant component), the weight ratio of thecharged, bilayer-associated compound to the liquid crystal should bebetween about 0.01:1 and 0.15:1, or more preferably between about 0.02:1and 0.08:1. If the charged compound is not a surfactant, it shouldpreferably be a liquid or at least a low-melting compound, that has ahigh partition coefficient, preferably greater than about 10, morepreferably greater than about 100, and most preferably greater thanabout 1,000. Uncoated particles are particularly useful when theparticles are used to capture specific compounds in the analyte (blood,urine, etc.) through either ionic interaction or biospecifically, by theincorporation of capture molecules in the cubic phase such asantibodies, receptors, complementary nucleic acids, chimeras, lectins,saccharides, etc. Likewise, specific interacting pairs such asantibody-antigen, receptor-ligand, RNA-RNA, avidin-biotin, etc., can beincorporated with one part of the pair in the liquid crystallineparticle and the other part immobilized on the SELDI chip.

A particularly useful class of embodiments of this invention includescases where capture molecules, for example antibodies, are incorporatedat the surface, and/or in the interior, of liquid crystalline particles,and the particles are added to analyte solution so as to capturespecific molecules of interest. In some cases, at least with the currentstate of art of mass spec-based detection of cancers, the molecule to becaptured will not actually be known, except for its molecular weight; insuch a case several capture molecules may be tried to determineempirically which is the best for capturing the most important analytemolecules.

Mass spec work to date has shown that there are apparently a handful ofcompounds, which seem to be peptide fragments, whose presence or level,taken in concert, in blood plasma are indicators of early-stage cancers.While one goal of SELDI-MS is to use the selectivity of SELDI chips toenhance the mass spec signal:noise ratio of these cancer indicators, itmay be unrealistic to think that the same SELDI chip can be selectivefor each one of these indicators. In the instant invention, the SELDIchip need only bind the particle of the invention, not the individualindicators. The job of binding the indicators can be delegated to theparticles of the invention, and indeed a single dispersion can containany number of different particles each selective for as few as oneindicator. Furthermore, the incorporation of the capture molecule (suchas receptor molecule, lectin, etc.) will in many cases be far superiorin a liquid crystalline particle, due to its lipid-based, biomimeticnature, its accessibility via continuous systems of nanopores, and bythe tremendous surface areas available for membrane proteins—which canbe hundreds of square meters per gram of cubic phase, in particular.Thus, it is entirely practical for a 10 microliter aqueous dispersion ofparticles of the instant invention to contain 0.1 to 1 square meter(1,000 to 10,000 sq. cm.) of internal surface area, accessible to amolecule with effective diameter less than that of the aqueous pores;this is in sharp contrast with surfaces areas on the order of 0.1 squarecentimeters for a simple SELDI chip spot. In addition, diffusion of amolecule into a particle of this invention in such a system will involvediffusional distances on the order of one micron, in contrast with 0.1to 1 millimeter for diffusion to a substrate.

Incorporating Capture Molecules.

Biomacromolecules, especially antibodies, which are particularly usefulas capture molecules in the practice of this invention can be selectedfrom the group of compounds that are listed as compatible with liquidcrystalline materials in U.S. Pat. No. 6,638,621. Generally speaking,concentrations of these reagents required for the assays of focus hereinare not very demanding, and are also sufficiently low that theincorporation of these compounds at the required levels will typicallyhave a minimal effect on the phase behavior of the lipid-based system.This being the case, a pre-existing composition, such as for a cubicphase with desirable properties, can simply be “pulled off the shelf”and the desired compound incorporated, without any particular danger ofchanging the phase or its properties significantly. The compound can beincorporated most preferably by simply dissolving it in one of thecomponents of the lyotropic liquid or liquid crystalline material, oftenthough not always the aqueous component, which will usually be loadedwith buffer components and/or salts, for stabilizing the compound. Insome cases, particularly where membrane proteins are involved, lipid andwater together will be required to solubilize the protein, but lipidsare inherent in the systems of discussion herein. If a membrane proteincomes supplied in a lipid-water (or sometimes glycerol) mixture,typically the lipids can be combined and found to be compatible withthose of the desired lyotropic composition (with the proper adjustment,of reducing the amount of added lipid in accounting for that already inthe protein preparation). In case of highly shear-sensitive compounds,the lyotropic particles can be first dispersed, and the protein or othercompound allowed to diffuse into the particles slowly over time underquiescent conditions.

In the case of capture molecules that do not partition strongly into thelyotropic material, this can be remedied by covalently bonding ahydrophobic anchor to the molecule, such as a palmitoyl or oleoyl chain.Compounds reactive to selected groups on proteins that would facilitatesuch a reaction are available commercially.

Reconstitutable Systems.

A crucial aspect of the embodiments of this invention is the stabilityof the compositions of the invention. When stored as particledispersions, the particles should be either substantially free fromcreaming and flocculation, or be such that creaming or flocculation areeasily reversed by simple shaking, prior to use. Reconstitutabledispersions can be of particular value in the instant invention.Spray-drying, freeze-drying, spray-congealing, electrospraying,supercritical fluid methods, and simple vacuum drying are among the waysof drying dispersions that can be applied in the practice of thisinvention. The appropriate stabilizers must be incorporated so thatthese powders form dispersions easily upon shaking by hand.

Dendrimers.

Dendrimers are highly-controlled MW compounds that can be used, insteadof peptides and proteins, as macromolecules (or “markers”) in thepractice of this invention. In one type of embodiment, one or moredendrimers is (are) dissolved or dispersed in particles of theinvention, and the particles (and therefore the dendrimer as well, byassociation) are then bound to the desired assay substrate, such as aSELDI chip. One possible complication with the use of dendrimers istheir tendency to flocculate proteins, so it is generally best to notinclude important proteins in the same particles as contain thedendrimers.

Polymerized Materials.

U.S. Pat. No. 5,244,799 (the contents of which are hereby incorporatedby reference in entirety) reports the polymerization of nanostructuredcubic and hexagonal phase liquid crystals, with retention of theirnanostructure. The retention of structure was demonstrated bysmall-angle x-ray scattering (SAXS) and transmission electron microscopy(TEM). The possibility of polymerizing the cubic phase of a particle ofthe instant invention opens up a number of possibilities, particularlyas they relate to increasing the stability of the reversed liquidcrystalline phase and modulating its interaction with proteins, othermacromolecules, and also with components of biological materials foranalysis. Furthermore, the retention of a bilayer-bound protein might beincreased tremendously by polymerization, particularly if polymerizationobviated any tendencies for poresize changes with changing conditions.And the presence of a more permanent, precisely-defined pore structure,with precisely tunable poresize, might make possible improvedpresentation, and/or sequestration of a protein from degradative orother enzymes by size-exclusion from the pores of the polymerizedmatrix.

EXAMPLES

The following examples illustrate various embodiments of the presentinvention but are not to be construed as limiting the invention.

Example 1

A reversed cubic phase containing the protein insulin was prepared byfirst dissolving 0.111 grams of egg yolk ovomucoids (Belovo SA, Inc,Belgium) in 2.112 gm of a 20 mM sodium acetate, 0.5% sodium chloride, pH4 buffer solution; the latter prepared by dissolving 0.272 gm of sodiumacetate (Spectrum Chemical, Gardena, Calif.) and 0.500 gm of sodiumchloride (EM Science, Gibbstown, N.J.) in 100 mL of distilled water andadjusting the pH to 4 with 1M hydrochloric acid (Sigma Chemical Company,St. Louis, Mo.). Next, 0.040 gm insulin from bovine pancreas (SigmaChemical Company, St. Louis, Mo.) was dissolved in the buffer solution,and 0.004 gm phenol (Fisher Scientific, Fair Lawn, N.J.) added. Finally,3.251 gm linalool (Aldrich Chemical Company, Milwaukee, Wis.), and 3.282gm of Pluronic P123 (BASF, Mount Olive, N.J.) were added. After thoroughmixing the material was optically isotropic and of high viscosity. Ofthis, 8.384 gm of cubic phase was combined in a 50 mL beaker with 21.002gm of a diethanolamine-NAT solution; the latter prepared by mixing 8.047gm of diethanolamine (Aldrich Chemical Company, Milwaukee, Wis.), 18.382gm of distilled water, and 11.245 gm of N-acetyl-DL-tryptophan (MPBiomedicals, Aurora, Ohio). The cubic phase/diethanolamine-NAT mixturewas dispersed first with a Homogenizer (Brinkmann Polytron PT3000) at29.5 k rpm for three minutes, then with a Microfluidizer Processor(Microfluidics M110L) at 18 k psi for 1.5 minutes. To the microfluidizerwas then added 1.902 gm of diethanolamine and 10.309 gm of a 25% wt/wtzinc, acetate solution. Microfluidizing at 18 k psi was continued for 15runs of 1.5 minutes each, and then 2 mL of hot (60° C.) 6% wt/wtsorbitan monopalmitate dispersion (Spectrum Chemical, Gardena, Calif.)and 2 mL of 15% wt/wt aqueous albumin solution (Sigma Chemical Company,St. Louis, Mo.) were added. Following four more runs in themicrofluidizer the dispersion was divided in half.

Twenty mL of dispersion was centrifuged at 13.6 k rpm for 1.5 hours, thesupernatant discarded, and the centrifugate reconstituted with 0.5%Tween 80/0.25% SDS solution in a volume equal to that of the discardedsupernatant. This reconstituted sample was again dispersed, first withthe homogenizer (15 k rpm for 3 minutes) and then with themicrofluidizer (18 k rpm for 6 runs of 1.5 minutes each) and finallyfiltered thru a 5 micron syringe filter. This sample was saved as“Lyotropic/I2D.”

The other half of the dispersion was allowed to sit for approximately 24hours undisturbed, then microfluidized at 18 k rpm for 4 runs of 1.5minutes each. Next, 5 mL of dispersion was placed into each of 4centrifuge tubes containing approximately 0.16 gm of GAC 830 ActivatedCarbon (Norit, Atlanta, Ga.) and the tubes were agitated at 75 rpm for15 minutes on a shaker (Lab-line Junior Orbit Shaker). Each tube wasthen centrifuged (Clay Adams compact physicians centrifuge) for 5minutes at 4800 rpm. The top phase was filtered thru a 5 micron syringefilter and saved as “Lyotropic/I2.”

A 10% solution of the cationic polymer Eudragit E100 was prepared bymixing 0.504 gm of Eudragit E100 (Rohm Pharma Polymers, Germany), 0.500gm of lactic acid (Johnson Matthey, Ward Hill, Mass.), and 4.013 gmdistilled water. A 10% solution of the cationic surfactantMyristyltrimethylammonium Bromide was prepared by dissolving 0.507 gm ofMyristyltrimethylammonium Bromide (Aldrich. Chemical Company, Milwaukee,Wis.) in 4.509 gm of hot distilled water. A 10% solution of the cationicsurfactant Hexadecyltrimethylammonium Bromide was prepared by dissolving0.501 gm of Hexadecyltrimethylammonium Bromide (Sigma Chemical Company,St. Louis, Mo.) in 4.498 gm of hot distilled water. A 10% dispersion ofthe anionic surfactant K-Emplex was prepared by mixing 0.502 gm ofK-Emplex (American Ingredients Co., Grandview, Mo.) and 4.505 gm ofdistilled water. The 10% K-Emplex dispersion was vortexed and heated to75° C.

In separate 8 mL test tubes, 0.5 mL of 10% Eudragit E100 solution wasadded to 4.50 gm of “Lyotropic/I2” and “Lyotropic/I2D.” Next, 1.0 mL of10% Myristyltrimethylammonium Bromide solution was added to 4.00 gm of“Lyotropic/I2” and “Lyotropic/I2D.” Then 1.0 mL of 10%Hexadecyltrimethylammonium Bromide solution was added to 4.00 gm of“Lyotropic/I2” and “Lyotropic/I2D.” Finally, 1.0 mL of hot 10% K. Emplexdispersion was added to 4.00 gm of “Lyotropic/I2” and “Lyotropic/I2D.”The mixtures were quickly vortexed and sonicated upon each individualaddition to disperse.

The samples were named as follows:

-   -   I2-KE: “I2” coated with K-Emplex (sodium stearoyl lactylate)    -   I2-E100: “I2” coated with Eudragit E100    -   I2-HEX: “12” coated with hexadecyltrimethylammonium bromide    -   I2-MYR: “12” coated with myristyltrimethylammonium bromide    -   I2D-KE: “12D” coated with K-Emplex (sodium stearoyl lactylate)    -   I2D-E100: “12D” coated with Eudragit E100    -   I2D-HEX: “12D” coated with hexadecyltrimethylammonium bromide    -   I2D-MYR: “12D” coated with myristyltrimethylammonium bromide        Zeta potentials, as measured with a Beckmann-Coulter Doppler        Electrophoretic Light Scattering Analyzer, were found to average        −6 mV for I2D-KE (viz., negative, as expected from the anionic        surfactant), and +18 mV for the I2D-MYR sample.

These samples were then used in the Example 2.

Example 2

Doubly-coated microparticle dispersions loaded with the fluorescent dyeRhodamine B base were prepared using the same procedure as in Example 1,then tested for binding to Ciphergen SELDI chips. In each case analiquot of the dispersion was placed on one SELDI chip spot, incubatedfor about 15 minutes, and then washed. Using a Reichert-lung Polyvarfluorescence microscope, the fluorescence intensity was recorded as anindicator of particle binding. Four types of Ciphergen SELDI chips weretested: NP20-8, CM10-8, H50-8, and Q-10. Each dispersion was diluted 1to 10 with Krebbs Ringer buffer (pH 7.4, 4% albumin). Samples werelabelled “KE”, “E100”, “HEX”, and “MYR” as follows, with the first andsecond coatings indicated:

-   -   KE) Zinc-NAT/K-Emplex (sodium stearoyl lactylate)    -   E100) Zinc-NAT/Eudragit E100    -   HEX) Zinc-NAT/hexadecyltrimethylammonium bromide    -   MYR) Zinc-NAT/myristyltrimethylammonium bromide        The results were as follows:    -   Q-10 chip: KE and E100 showed very intense fluorescence, much        weaker for MYR and HEX; E100>KE>>HEX=MYR.    -   NP20-8 chip: nothing was very intense or well covered throughout        the spot, but KE had some fluorescence; KE>>E100>MYR>HEX.    -   CM10-8 chip: HEX and MYR had very intense fluorescence, good        spot coverage with defined edges; HEX>MYR>>KE>E100.    -   H50-8 chip: E100 and KE showed some fluorescence;    -   E100>KE>>HEX>MYR.        The results indicate that strong binding can be achieved when        the surface charge of the microparticle is made opposite in sign        to the charge on the chip. Thus, the anionic surfactant-coated        particles in sample “KE” bound strongly to the quaternary        ammonium functionalized “Q-10” chip, and the cationic        surfactant-coated particles in samples “MYR” and “HEX” bound        strongly to the carboxylated “CM10-8” chip. The latter result in        particular indicates the utility of the double-coating approach,        since the particle surface prior to the second coating is        negative (approximately −10 mV zeta potential for the zinc-NAT        coated particles). It is also important to note that        singly-coated (zinc-NAT only) particles bound moderately to the        cationic Q-10 chips, due to their mildly anionic zeta potential.

Example 3

An L2 phase containing the anesthetic propofol was first prepared in a100 mL test tube by mixing 0.302 grams of propofol (AlbemarleCorporation, Baton Rouge, La.), 0.272 gm of vitamin E (Archer DanielsMidland Co., Decatur Ill.), and 1.653 gm of Pluronic L122 (EthoxChemicals, Greenville, S.C.). Next, 0.162 gm of the anionic surfactantsodium deoxycholate (Aldrich Chemical Company, Milwaukee, Wis.) and0.487 gm of glycine (Spectrum Chemical Company, Gardena, Calif.) weredissolved in 29.352 gm of distilled water. Then, 27.797 gm of thesurfactant solution was added to the 100 mL test tube containing thepropofol L2 phase. Upon contact with water, a reversed cubic phase wasformed and subsequently dispersed using a homogenizer (Brinkman PolytronPT 3000) at 29 k rpm for 10 minutes. The dispersion was filtered using a0.22 μm PVDF syringe filter (Millipore, Ireland). Observation in aReichert-Jung Polyvar microscope operating in differential interferencecontrast (DIC) mode demonstrated that a particle size on the order of200 nanometers had been achieved. This dispersion of uncoated particleswas referred to as PF1112304 L2.

Example 4

The components of a reversed cubic phase containing the local anestheticbupivacaine were combined in a 50 mL test tube by first dissolving 0.454grams of free base bupivacaine in 1.829 gm of Vitamin E (Archer DanielsMidland Co, Decatur Ill.) and heating to 60° C. The free basebupivacaine was prepared by dissolving 25.019 gm of bupivacaine HClmonohydrate (Spectrum Chemical Company, Gardena, Calif.) in 600 mL ofdistilled water, then adding a 70 mL of 1.0N NaOH (Spectrum ChemicalCompany, Gardena, Calif.), decanting off the liquid, and drying the freebase bupivacaine using a RotoVap (Polyscience, Niles, Ill.) with 30 mBarvacuum (BrandTech Scientific, Essex, Conn.) applied for 4 hours.Following dissolution of bupivacaine in vitamin E, 0.919 gm of sterilewater and 1.831 gm of warm (40° C.) Pluronic P123 (BASF, Greenville,S.C.) were added to the test tube. Next, 12.445 gm of adiethanolamine-NAT solution was added to the 50 mL test tube containingthe cubic phase components. The diethanolamine-NAT solution was preparedby mixing 3.148 gm of diethanolamine (Aldrich Chemical Company,Milwaukee, Wis.), 7.364 gm of distilled water, and 4.505 gm ofN-acetyl-DL-tryptophan (MP Biomedicals, Aurora, Ohio). The cubicphase/diethanolamine-NAT mixture was homogenized (Brinkmann PolytronPT3000) at 29.5 k rpm for two minutes. While the material was beinghomogenized, 0.643 gm of diethanolamine and 9.489 gm of a 16.6% wt/wtzinc acetate solution (Sigma Chemical Company, St. Louis, Mo.) wereadded. Homogenizing continued for five minutes, and then the mixture wasquickly transferred to a microfluidizer (Microfluidics M110 L) where 8runs of 1.5 minutes each at 18 kspi were performed. Next, 1.1 mL of hot(60° C.) 6% wt/wt sorbitan monopalmitate dispersion (Spectrum Chemical,Gardena, Calif.) and 1.1 mL of 15% wt/wt aqueous albumin solution (SigmaChemical Company, St. Louis, Mo.) were added while microfluidizing.Following 4 more microfluidizing runs of 1.5 minutes each, thedispersion was pumped out and allowed to sit overnight. The followingday, 4 more microfluidizing runs of 1.5 minutes were completed. Thedispersion was then divided into 6 centrifuge tubes of 3.5 mL ofdispersion each. Approximately 0.14 gm of GAC 830 Activated Carbon(Norit, Atlanta, Ga.) was added to each tube and the tubes were agitatedat 100 rpm for 15 minutes on a shaker (Lab-line Junior Orbit Shaker).Each tube was then centrifuged (Clay Adams compact physicianscentrifuge) for 5 minutes at 4800 rpm. The top phase was pipetted offand filtered using a 5 μm PVDF syringe filter (Millipore, Ireland). Thisdispersion was labelled “F2V102604 top phase”.

An important aspect of this Example is the fact that uncoated particleswere converted to coated particles (coated by the zinc salt of NAT) bythe addition of zinc acetate.

Example 5

A reversed cubic phase was prepared by first dissolving 0.040 grams ofegg yolk ovomucoids (Belovo SA, Inc, Belgium) in 1.036 gm of a 20 mMsodium acetate, 0.5% sodium chloride, pH 4 buffer solution; the latterprepared by dissolving 0.272 gm of sodium acetate (Spectrum Chemical,Gardena, Calif.) and 0.500 gm of sodium chloride (EM Science, Gibbstown,N.J.) in 100 mL of distilled water and adjusting the pH to 4 with 1Mhydrochloric acid (Sigma Chemical Company, St. Louis, Mo.). Next, 1.504gm Vitamin E (Archer Daniels Midland Co, Decatur Ill.) and 2.399 gm ofPluronic L122 (Ethox Chemicals, Greenville, S.C.) were added. Afterthorough mixing, the material was optically isotropic and of highviscosity. Of this, 4.005 gm of cubic phase was combined in a 100 mLtest tube with 10.049 gm of a diethanolamine-NAT; the latter prepared bymixing 5.248 gm of diethanolamine (Aldrich Chemical Company, Milwaukee,Wis.), 12.273 gm of distilled water, and 7.505 gm ofN-acetyl-DL-tryptophan (MP Biomedicals, Aurora, Ohio). The cubicphase/diethanolamine-NAT mixture was first vortexed, then homogenized(Brinkman Polytron PT3000) at 29.5 k rpm for two minutes. While thematerial was being homogenized, 0.553 gm of diethanolamine and 7.547 gmof a 16.6% wt/wt zinc acetate solution were added. Homogenizingcontinued for five minutes, and then 0.8 mL of hot (60° C.) 6% wt/wtso/titan monopalmitate dispersion (Spectrum Chemical, Gardena, Calif.)and 0.8 mL of 15% wt/wt aqueous albumin solution (Sigma ChemicalCompany, St. Louis, Mo.) were added. Following five more minutes ofhomogenizing, the dispersion was divided into 6 centrifuge tubes of 3.5mL of dispersion each. Approximately 0.14 gm of GAC 830 Activated Carbon(Norit, Atlanta, Ga.) was added to each tube and the tubes were agitatedat 100 rpm for 15 minutes on a shaker (Lab-line Junior Orbit Shaker).Each tube was then centrifuged (Clay Adams compact physicianscentrifuge) for 5 minutes at 4800 rpm. The top phase was pipetted offand saved as “Lyotropic/IC2 BLANK.”

A 10% solution of the cationic polymer Eudragit E100 was prepared bymixing 0.504 gm of Eudragit E100 (Rohm Pharma Polymers, Germany), 0.500gm of lactic acid (Johnson Matthey, Ward Hill, Mass.), and of 4.013 gmdistilled water. A 10% dispersion of the anionic surfactant K. Emplexwas prepared by mixing 0.5016 gm of K. Emplex (American Ingredients Co.,Grandview, Mo.) and 4.505 gm of distilled water. The 10% K. Emplexdispersion was vortexed and heated to 75° C. In separate 8 mL testtubes, 0.5/mL of 10% Eudragit E100 solution was added to 4.502 gm of“Lyotropic/IC2 BLANK” and 1.0 mL of hot 10% K; the result was named“IC2102604 1% eudragit E100”. Emplex dispersion was added to 4.002 gm of“Lyotropic/IC2 BLANK”, and the resulting dispersion named “IC2102604 2%K Emplex” (or “IC2-KE”). The mixtures were immediately vortexed todisperse.

Example 6

In this experiment, various embodiments of the current invention wereused as blocking agents in an ELISA experiment, to determine the degreeto which the particles bound to the substrate and blocked the adsorptionof an antibody.

Reagents and materials used:

1. Phosphate-buffered saline (PBS): 150 mM NaCl, 150 mM Na₂HPO₄/NaH₂PO₄pH 7.2

2. BSA: 1% bovine serum albumin (Sigma A7906) in PBS

3. Ab-HRP conjugate: monoclonal anti-goat IgG—peroxidase (Sigma A9452,approx 6.5 mg/ml) diluted 1:10,000 in PBS

4. Stop soln: 0.5 M H₂SO₄

5. TMB: Sigma T8665, a peroxidase substrate solution based ontetramethylbenzidine

6. ELISA plates: Nalge Nunc 96 well high flange, 300 ul capacity,uncoated polystyrene

Four embodiments of the current invention were used:

L1: F2V102804 prepared as described in Example 4

L2: PF1112304 L2 prepared as described in Example 3

L3: IC2102604 1% eudragit E100 prepared as described in Example 5

L4: IC2102604 2% K emplex prepared as described in Example 5

The procedure used was as follows.

1. Into an ELISA well, pipette 300 ul of preparation & incubate 10minutes at room temperature:

well content 1-4 PBS 5-8 BSA  9-12 L1 13-16 L2 17-19 L3 21-23 L42. Aspirate well contents, add 300 ul PBS, aspirate contents & blot dry.3. Add to each well 50 ul Ab-HRP conjugate. Incubate 20 minutes at roomtemperature.4. Aspirate well contents followed by 2×300 ul PBS rinses. Blot dry.5. Add 50 ul TMB, incubate 10 minutes room temperature.6. Add 50 ul Stop solution.7. Photograph ELISA plate.8. Combine contents of replicate wells and dilute 1:10 with 50% PBS/50%Stop soln.Measure A450 (absorbance at 450 nm) versus PBS blank.

Results. The results are shown in the following chart:

Blocking Preparation A450 PBS 1.9630 BSA 0.2420 F2V102804 top phase0.0680 PF1112304 L2 0.2090 IC2102604 1% eudragit E100 0.0570 IC21026042% K emplex 0.0510Thus, three coated particle preparations of the instantinvention—IC2102604 1% eudragit, IC2102604 2% K-Emplex, bothdoubly-coated, and the singly-coated F2V 102804—exhibited greaterpolystyrene blocking capability than the standard blocking preparationof 1% bovine serum albumin. Preparation PF1112304 of uncoated particlesexhibited slightly better blocking than BSA.

Example 7

The K-Emplex-coated (anionic surface) particle sample, here termed“I2-KE”, produced in Example 1, and thus loaded with insulin, wasselected for SELDI experiments using positively-charged “Q-10” chips.The procedure to prepare the SELDI chips in all the Examples reportedherein was as follows, with Steps 2-6 accomplished by the robotics.

Step 1: Dilute the particle dispersion in 50 mM buffer, pH 7.4;

Step 2: Pretreat chips with buffer, 5 minutes×2;

Step 3: Add 25 ul of diluted sample to each Bioprocessor well;

Incubate 30 minutes at room temperature;

Step 4: Wash chips 4× with 150 ul of buffer with 10 mixing cycles;

Wash chips 1× with water;

Step 5: Remove Bioprocessor. Air dry chips for 10 minutes;

Step 6: Add (microliter of SPA matrix in 50% acetonitrile/water 0.5%TFA;

Air dry for 15 minutes;

Repeat matrix application;

Air dry for 15 minutes before reading.

In this case, the “12-KE” dispersion was diluted by a factor of 1:1000,in Step 1. The chips were analyzed in a Ciphergen SELDI-MS instrument.

Results.

The following table shows the standard deviation, and the coefficient ofvariation, for each peak registered, before and after normalizing them/z ratio based on the insulin standard. The results are depictedgraphically in FIG. 5.

m/z Before After Mean SD % CV Mean SD % CV 3450.331 1.428905 0.041%3450.331 0.543094 0.016% 3899.16 1.838274 0.047% 3899.16 0.850905 0.022%4160.347 1.943068 0.047% 4160.347 0.416087 0.010% 4187.138 3.8661380.092% 4187.139 5.216812 0.125% 4255.586 2.16548 0.051% 4255.5861.222404 0.029% 4364.707 1.869249 0.043% 4364.707 0.275111 0.006%4476.072 1.933504 0.043% 4476.072 0.163337 0.004% 4634.313 2.0278740.044% 4634.313 0.187323 0.004% 5743.398 2.565501 0.045% 5743.398 00.000% 6235.33 3.024563 0.049% 6235.33 0.460464 0.007% 6442.225 3.026080.047% 6442.225 0.273127 0.004% 6640.382 3.047743 0.046% 6640.3820.204372 0.003% 6890.079 3.366266 0.049% 6890.079 0.418004 0.006%6930.184 3.417675 0.049% 6930.184 0.395398 0.006% 6949.548 3.5359250.051% 6950.034 0.366776 0.005% 7625.439 3.641834 0.048% 7625.4390.407421 0.005% 7774.885 3.819105 0.049% 7774.884 0.435893 0.006%8214.542 4.178585 0.051% 8214.072 0.587112 0.007% 8573 4.139301 0.048%8573 0.678145 0.008% 8700.976 4.334909 0.050% 8700.976 0.669145 0.008%8778.434 4.622205 0.053% 8778.433 0.887131 0.010% 8924.732 4.5484310.051% 8924.731 0.768855 0.009% 9140.543 4.652185 0.051% 9140.5430.755525 0.008% 9430.05 4.863498 0.052% 9430.05 0.839832 0.009% 9649.3385.012949 0.052% 9649.338 2.016245 0.021% 9719.936 5.048652 0.052%9719.936 0.905098 0.009% 12460.17 6.748364 0.054% 12460.17 1.5239450.012% 12616.58 6.768427 0.054% 12616.58 1.475958 0.012% 13769.237.617153 0.055% 13769.23 2.132805 0.015% 13855.5 6.067183 0.044%13854.68 1.070892 0.008% 13884.03 7.195736 0.052% 13886.74 2.1878850.016% 15137.36 8.869256 0.059% 15137.36 2.554225 0.017% 15878.589.328144 0.059% 15878.58 3.144241 0.020% 17266.12 8.3904 0.049% 17266.122.886141 0.017% 17393.28 9.245409 0.053% 17393.28 2.697426 0.016%17895.74 14.335 0.080% 17898.42 13.67282 0.076% 21509.6 6.305882 0.029%21511.43 5.648626 0.026% 21776.09 8.866126 0.041% 21780.64 4.2793690.020% 28067.07 14.04885 0.050% 28069.45 6.282196 0.022% 5.171893 0.051%1.782055 0.016%As can be seen, the standard deviation in peak position, calculated fromeight repetitions, was reduced more than three-fold, from 5.17 (0.051%of mean) to 1.78 (0.016% of mean) by the use of this encapsulatedinsulin calibrant.

It should be noted that SELDI peaks registered for insulin in this, andother, experiments with the instant invention were in all cases ofextremely high intensity, allowing very high dilutions (1:5000 and more)with the maintenance of a strong, sharp signal from the insulin. Thisindicates a strong propensity of the particles to achieve a high rate ofdeposition of the insulin onto the Q-10 chips, as well as othersubstrata.

Furthermore, the dispersion in this Example was tested for encapsulationof the insulin, in the following way. A centricon centrifuge filter wasused to remove the particles from the aqueous exterior phase, and thelatter was tested for insulin (un-encapsulated, or “free” insulin),using a standard ELISA assay. No free insulin was detected in thisexperiment, demonstrating a complete encapsulation of the peptide, anindication of the very high partition coefficient in the cubic phaseinterior over water.

Example 8

A dispersion of uncoated, anionically-charged cubic phase microparticleswas first prepared. A reversed cubic phase containing AntiMouse IgG wasprepared in an 8 mL test tube by combining 0.099 gm AntiMouse IgG (SigmaChemical Company, St. Louis, Mo.) along with 0.229 gm sterile water(Abbott Labs, North Chicago, Ill.) and 0.470 gm vitamin E (ArcherDaniels Midland Company, Decatur, Ill.). Lastly, 0.733 gm of PluronicL122 (Ethox Chemicals, Greenville, S.C.) was added, and after thoroughmixing the material was optically isotropic and of high viscosity. Ofthis, 1.220 gm of cubic phase was added to a 50 mL beaker into which hadpreviously been dissolved 0.061 gm of deoxycholic acid, sodium salt(Aldrich Chemical Company, Milwaukee, Wis.) and 0.272 gm glycine(Spectrum Chemical, Gardena, Calif.) into 16.484 gm of sterile water.The cubic phase/aqueous solution was dispersed first with a Homogenizer(Brinkmann Polytron PT3000) at 29.5 k rpm for one minute, then with aMicrofluidizer Processor (Microfluidics M110L) at 18 k psi for two runsof 1.5 minutes each. The sample was removed from the Microfluidizer andthe pH measured at 7.4 (Hanna Instruments, Woonsocket, R.I.) beforefiltering through a 0.22 um PVDF syringe filter (Millipore Corporation,Bedford, Mass.). The sample was denoted “Lyotropic/AM1”, oralternatively as “AM1 Lyocells”.

This dispersion of uncoated cubic phase particles, which was denoted“AM1 LyoCells”, was then tested for its effect on SELDI analysis ofblood proteins. A 1:1000 dilution of this dispersion was added to serumfrom a normal (cancer-free) patient population, and the spiked plasmaanalyzed with a Ciphergen Q-star SELDI system employing cationic(anion-exchange) “Q-10” chips. The resulting mass spec data are showngraphically in FIG. 5; and numerically in the following table:

Mean m/z Buffer % CV I1 % CV AM1 % CV 3083.79 1.167 82.2% — — 3443.055.337 28.2% — — 3485.60 2.387 25.9% — — 3815.55 1.689 63.4% — — 3891.462.321 28.3% 3.304 13.7% 4.313 6.1% 4151.86 41.087 5.4% 49.931 8.6%63.588 7.4% 4184.28 13.421 25.3% 19.334 12.9% 25.335 8.3% 4245.32 3.95840.5% 5.129 7.9% 6.658 9.3% 4357.41 3.830 24.7% 4.929 8.0% 6.704 13.6%4467.30 4.022 23.4% 5.005 9.5% 6.859 10.3% 4625.28 4.735 34.5% 5.7487.3% 7.921 8.2% 4708.90 3.626 4.7% 4.233 8.1% 5736.28 2.579 5.9% 6224.721.347 62.2% 2.010 19.6% 2.150 20.4% 6431.59 5.071 30.6% 16.521 6.6%21.157 8.2% 6629.53 11.053 32.5% 29.316 5.9% 37.997 7.8% 6832.91 4.7249.5% 6879.07 5.713 38.9% 8.691 7.6% 11.094 5.7% 6939.00 7.591 36.1%10.250 9.8% 12.738 5.1% 7613.94 4.216 20.1% 4.886 8.0% 6.354 6.1%7763.54 4.078 19.7% 3.275 58.7% 3.126 18.1% 8201.88 0.920 11.5% 2.4458.4% 3.314 12.1% 8560.28 2.337 11.2% 3.563 4.0% 8687.14 2.195 31.1%5.758 5.0% 8.875 6.4% 8762.00 4.404 6.3% 6.285 6.7% 8807.26 1.853 44.0%4.903 1.4% 7.103 6.4% 8911.83 4.259 7.9% 11.931 7.8% 16.922 7.6% 9127.653.330 19.9% 11.524 6.3% 16.290 9.3% 9298.99 3.281 10.0% 9416.53 7.00119.0% 23.128 6.4% 30.925 6.6% 9636.71 1.642 9.2% 4.583 8.0% 6.051 3.5%9706.07 2.719 17.5% 8.371 8.9% 10.822 6.9% 9926.52 1.865 8.3% 10066.30.782 27.8% 10640.5 0.432 35.9% 12444.4 5.049 56.5% 6.970 15.1% 9.02214.4% 12600.7 2.115 52.4% 2.757 12.5% 3.559 15.1% 12833.9 0.763 40.1%1.045 8.2% 1.487 2.6% 13750.6 12.483 44.9% 17.964 2.6% 24.818 3.4%13872.1 15.965 39.6% 20.797 3.2% 28.968 6.1% 14046.7 4.826 30.7% 6.2170.9% 8.522 5.2% 15116.4 0.622 8.1% 0.606 14.4% 0.716 7.1% 15856.1 0.32817.1% 0.310 14.1% 0.391 12.3% 17125.8 1.108 8.9% 1.839 11.9% 17244.91.002 26.4% 2.364 9.3% 4.042 12.6% 17371.2 1.030 26.3% 2.442 8.1% 4.11612.6% 17862.9 0.268 17.0% 0.615 7.9% 1.019 3.6% 18315.8 0.314 8.3% 0.8486.9% 18602.4 0.499 13.0% 21025.0 0.323 11.1% 21485.2 0.265 21.0% 0.2674.4% 0.364 18.5% 21754.4 0.276 13.1% 0.239 6.0% 0.333 21.6% 22256.90.272 81.7% 23174.5 0.287 7.7% 0.420 24.3% 26301.6 0.061 29.3% 28038.01.425 25.8% 2.288 8.9% 3.858 22.4% 28825.0 0.829 28.8% 31091.3 0.1672.6% 0.230 12.7% 33340.7 1.183 103.0% 0.293 25.1% 33528.8 0.233 11.7%0.280 26.2% 33838.1 0.794 84.2% 0.230 10.3% 33946.7 0.230 10.3% 37019.30.116 34.5% 37245.5 0.122 16.6% 37392.2 0.071 7.7% 0.127 22.9% 38713.50.106 42.3% 0.125 19.6% 0.184 38.1%

Several conclusions can be drawn from the FIG. 5. First, intensities areclearly and consistently increased in the presence of cubic phaseparticle dispersion AM1 (and to a lesser extent with another cubic phasepreparation, “I1”), as is the signal-to-noise ratio, and this is truefor essentially every peak registered. Secondly, some proteins that werenot registered in the absence of the particles, were detected in thepresence of the AM1 LyoCell particles. For example, a moderately strongpeak at about m/z=6833 is clearly registered with “AM1” particlespresent, even though it is not detectable without the particles present.Obviously the cubic phase particles are binding well to the Q-10 chips,presumably by virtue of their anionic charge, with the implication thatany proteins absorbed by, or adsorbed to, the particles will be broughtdown to the surface—in some cases, at least, when they would notnormally bind to the substrate. And third, the coefficient of variation(CV) is dramatically lower in the presence of the cubic phasedispersions than in the absence thereof. This strong effect ofdecreasing the variability of the measurement is not simply thenon-specific effect of surfactant, since it was not seen when Triton-Xwas added in place of the cubic phase particles.

Notwithstanding the appearance of new peaks, the overall spectra withand without particles are very similar with the main effect beingamplification of the signal, and greatly reducing the variability. It isquite likely that the particles are bringing down to the substrate arich concentration of proteins, increasing the number of anionicproteins reaching (and binding to) the substrate, but that proteinswhich are cationic at this pH nevertheless desorb in the course of theexperiment, and new peaks which appear in the presence of the particlesare most likely those which are uncharged or weakly charged (that is,near their isoelectric point). Tentatively at least, one can draw thefollowing conclusions from these data:

-   -   1) these particles can significantly improve the signal strength        and signal-to-noise ratio of the registered peaks;    -   2) for the most part, the selectivity obtained by the use of a        SELDI substrate is retained, in the presence of the particles;    -   3) likely, it is possible to identify proteins near their        isoelectric point, by noting those peaks that appear in the        presence of the particles but not in their absence.

Example 9

A dispersion of doubly-coated particles loaded with both bovine insulinand beta-casein, with an outer coating of Eudragit E100, was firstprepared as follows. A reversed cubic phase containing the proteinsinsulin and beta-casein was prepared by first dissolving 0.048 grams ofegg yolk phospholipids (Belovo SA, Inc, Belgium) in 1.251 gm of a 20 mMsodium acetate, 0.5% sodium chloride, pH 4 buffer solution; the latterprepared by dissolving 0.272 gm of sodium acetate (Spectrum Chemical,Gardena, Calif.) and 0.500 gm of sodium chloride (EM Science, Gibbstown,N.J.) in 100 mL of distilled water and adjusting the pH to 4 with 1Mhydrochloric acid (Sigma Chemical Company, St. Louis, Mo.). Next, 0.016gm insulin from bovine pancreas and 0.015 gm beta-casein (both fromSigma Chemical Company, St. Louis, Mo.) were added to the buffersolution, and 0.001 gm rhodamine B base (Aldrich Chemical Company,Milwaukee, Wis.) added. Finally, 1.803 gm vitamin E (Archer DanielsMidland Company, Decatur, Ill.), and 2.888 gm of Pluronic L122 (EthoxChemicals, Greenville, S.C.) were added. After thorough mixing thematerial was optically isotropic and of high viscosity. Of this, 4.999gm of cubic phase was combined in a 50 mL beaker with 12.478 gm of adiethanolamine-NAT solution; the latter prepared by mixing 3.151 gm ofdiethanolamine (Aldrich Chemical Company, Milwaukee, Wis.), 7.358 gm ofdistilled water, and 4.503 gm of N-acetyl-DL-tryptophan (MP Biomedicals,Aurora, Ohio). The cubic phase/diethanolamine-NAT mixture was dispersedwith a Homogenizer (Brinkmann Polytron PT3000) at 29.5 k rpm for threeminutes. To the homogenizer was then added 0.599 gm of diethanolamineand 9.441 gm of a 16.6% wt/wt zinc acetate solution. Homogenizing at29.5 k rpm was continued for five minutes, and then 1.1 mL of hot (60°C.) 6% wt/wt sorbitan monopalmitate dispersion (Spectrum Chemical,Gardena, Calif.) and 1.1 mL of 15% wt/wt aqueous albumin solution (SigmaChemical Company, St. Louis, Mo.) were added. Following five additionalminutes of homogenizing, 4.5 mL of dispersion was placed into each of 6centrifuge tubes containing approximately 0.14 gm of GAC 830 ActivatedCarbon (Norit, Atlanta, Ga.) and the tubes were agitated at 100 rpm for15 minutes on a shaker (Lab-line Junior Orbit Shaker). Each tube wasthen centrifuged (Clay Adams compact physicians centrifuge) for 5minutes at 4800 rpm. The top phase was saved as “Lyotropic/IC2.”

This dispersion was then tested in a SELDI analysis of blood plasmaproteins, employing the cationic “Q-10” chips from Ciphergen. Insulin,with a MW of approximately 5743.5 and beta-casein at approximately24,075 were both registered even when the dispersion was diluted by1:5000 in Step 1 of the procedure described above. Averaging two runs ateach of 4 dilutions, the intensities of these two peaks were as follows:

Dilution 1:500 1:1000 1:2500 1:5000 Insulin intensity 40.4 17.0 7.1 5.3Casein intensity 0.08 0.03 0.02 0.03Thus, a single particle loaded with two proteins has yielded two peaks,corresponding to the two proteins, in this SELDI-MS experiment.

Example 10

Antibody was incorporated in uncoated cubic phase particles in thisExample and shown to bind to an ELISA plate, and subsequently to bind asecond antibody much more strongly than control particles without thefirst antibody. The experiment also demonstrates the strong NSB-blockingproperty of the particles.

A reversed cubic phase containing AntiMouse IgG was prepared in an 8 mLtest tube by combining 0.055 gm AntiMouse IgG (Sigma Chemical Company,St. Louis, Mo.) along with 0.662 gm Patchouli Oil (Aura Cacia, Norway,Iowa), 0.080 gm dimyristoyl phosphatidylglycerol (NOF, Tokyo, Japan) and0.434 gm of a 6% deoxycholic acid, sodium salt (Aldrich ChemicalCompany, Milwaukee, Wis.) solution. Lastly, 0.822 gm ofphosphatidylcholine (Avanti Polar Lipids, Alabaster, Ala.) was added,and after thorough mixing the material was optically isotropic and ofhigh viscosity. Of this, 1.366 gm of cubic phase was added to a 50 mLbeaker into which had previously been dissolved 0.101 gm glycine(Spectrum Chemical, Gardena, Calif.) into 18.512 gm of distilled water.Three drops of 1N NaOH (Spectrum Chemical, Gardena, Calif.) were addedbefore the cubic phase/aqueous solution was dispersed, first with aHomogenizer (Brinkmann Polytron PT3000) at 29.5 k rpm for one minute,then with a Microfluidizer Processor (Microfluidics M110L) at 18 k psifor two runs of 1.5 minutes each. The sample was removed from theMicrofluidizer and the pH measured at 7.8 (Hanna Instruments,Woonsocket, R.I.) before filtering through a 0.45 um PVDF syringe filter(Millipore Corporation, Bedford, Mass.). The sample was saved as“Lyotropic/AM5011405.” Similar samples with no protein, and with albuminin place of the antibody, were also prepared.

The reagents and substrate used in the experiment were as follows:

1. Antibody1: rabbit anti-mouse IgG (Sigma M7023, Lot 083K4837, 2.8mg/ml) 1:5,000 in PBS.

2. Antibody2: mouse anti-goat IgG-HRP conj (Sigma A9452, Lot 023K4819,6.5 mg/ml) 1:5,000 in PBS.

3. Tetramethylbenzidine (TMB): Sigma T8665.

4. Stop soln: 0.5N sulfuric acid.

5. ELISA plates: Nalge NUNC 96 well high plane, uncoated polystyrene 300μl capacity.

6. PBS: 0.01M phosphate, 0.138 M NaCl 0.0027M KCl pH7.4 Sigma P3813).

Uncoated cubic phase particle preparations, with L1 and L2 being theblanks, and L3 the “live” antibody-containing dispersion:

L1: blank, uncoated, phosphatidylcholine/patchouli/deoxycholate, noprotein (AM5012005).

L2: blank, uncoated, PC/patchouli/deoxycholate with albumin at 2.3 μg/ml(AM5012005).

L3: blank, uncoated, PC/patchouli/deoxycholate with antibody1 at 4.6μg/ml (AM5011405).

Procedure:

1. Pipette 300 μL PBS or cubic phase preparation into quadruplicateELISA wells, incubate 20 minutes at RT, according to the following(herein the term “lyocells” refers to a dispersion of cubic phaseparticles):

Sample Contents Comments C0 PBS control C1 L1 blank lyocell C2 L2lyocell + albumin S1 L3 lyocell + Antibody1 S2 L3/C1 1:10 dilution of S1into buffer S3 L3/C1 1:10 dilution of S2 into buffer S4 L3/C1 1:10dilution of S3 into buffer S5 L3/C2 1:10 dilution of S1 into blanklyocells S6 L3/C2 1:10 dilution of S5 into blank lyocells S7 L3/C2 1:10dilution of S6 into blank lyocells S1-3X L3 S1 washed 3× at step 2 S2-3XL3/C1 S2 washed 3× at step 2 S3-3X L3/C1 S3 washed 3× at step 2 S4-3XL3/C1 S4 washed 3× at step 2 S5-3X L3/C2 S5 washed 3× at step 2 S6-3XL3/C2 S6 washed 3× at step 2 S7-3X L3/C2 S7 washed 3× at step 22. Aspirate well contents & wash w/300 μL PBS & blot3. Pipette 50 μL Antibody2 (conjugate). Incubate 30 min RT4. Aspirate well contents & wash w/300 μL PBS & blot5. Pipette 100 μL TMB solution & incubate 15 min RT6. Pipette 200 μL stop solution.7. Combine 200 μl of 2 representative wells and read A450

Results:

Sample Description A450 C0 PBS buffer >3.0 C1 naked lyocells − noprotein 0.188 C2 naked lyocells + albumin 0.213 S1 naked lyocell +antibody 0.655 S2 S1 diluted 1:10 w/PBS 0.245 S3 S2 diluted 1:10 w/PBS0.546 S4 S3 diluted 1:10 w/PBS >3.0 S5 S1 diluted 1:10 w/C2 0.308 S6 S5diluted 1:10 w/C2 0.187 S7 S6 diluted 1:10 w/C2 0.183 S1-3X S1 w/3× wash0.484 S2-3X S2 w/3× wash 0.211 S3-3X S3 w/3× wash 0.452 S4-3X S4 w/3×wash >3.0 S5-3X S5 w/3× wash 0.228 S6-3X S6 w/3× wash 0.172 S7-3X S7w/3× wash 0.192

Thus, the antibody-containing lyocells demonstrated more capacity tobind the conjugate that either the blank or albumin-containing lyocells.In addition, all preparations showed very strong NSB-blocking capacityrelative to buffer.

Serial dilution of antibody-containing lyocell with blank lyocellsdemonstrated a dose-response. If the lyocell-coated wells were washedthree times instead of once (before conjugate is added), some of thebinding capacity was eliminated.

Example 11

In this Example, a preparation of insulin-containing cubic phaseparticles as described above was shown to deposit insulin effectively ona traditional ELISA substrate, and the insulin was then detected in anELISA assay.

Reagents Used:

Antibodies (all dilutions made with PBS):

anti-hCG monoclonal (Fitzgerald clone M94138) as a Control

diluted 1:200 (final conc.=5 μg/mL)

and 1:2000 (final conc.=0.5 μg/mL)

anti-Insulin monoclonal from Abcam (clone ab7760)

diluted 1:100 (final conc=1 μg/mL)

diluted 1:1000 (final conc.=0.1 μg/mL)

Protocol

E100 doubly-coated cubic phase particles containing Insulin were diluted1/10 in Phosphate Buffer (pH 7.6), and in Carbonate Buffer (pH 8.5). Allwells coated overnight at 4° C. (100 μL/well), then rinsed with PBS thenblocked with SuperBlock 1 hour (300 μL/well), and again rinsed 3× withPBS. Mouse anti-insulin monoclonal antibodies added and incubated for 1hour (100 μL/well). The wells were then rinsed 5× with PBS. Goatanti-mouse HRP conjugate was then added (1:2500 dilution) incubated for1 hour (100 μL/well), after which the wells were rinsed 5× with PBS. HRPsubstrate was finally added (100 μL/well), and the reaction stopped with0.1 M HCl (100 μL/well).Results.

The optical densities (OD) in the wells read as follows:

Phosphate Carbonate Buffer Buffer OD 450 Mean OD 450 Mean Blank 0.0550.055 0.054 0.053 0.054 0.051 anti-hCG (500 ng) 0.060 0.067 0.063 0.0650.074 0.066 anti-hCG (50 ng) 0.061 0.058 0.061 0.070 0.054 0.078anti-Insulin (100 ng) 0.715 0.743 0.710 0.723 0.761 0.735 0.754 0.725anti-Insulin (10 ng) 0.705 0.705 0.676 0.687 0.701 0.697 0.709 0.687The high optical densities in the anti-insulin cases show that theinsulin was successfully deposited and accessible on the substrate, andthe low OD in the anti-HCG control shows that the binding was specificfor insulin/anti-insulin binding.

Example 12

A reversed cubic phase was prepared by combining 1.005 gm deionizedwater (Spectrum Chemical, Gardena, Calif.), 1.000 gm Carvone (AldrichChemical Company, Milwaukee, Wis.), 0:251 gm Strawberry Aldehyde (PentaManufacturing, Livingston, N.J.), 0.257 gm Sandalwood Oil (Cedar Vale,Cedar Vale, Kans.) and 2.509 gm of Pluronic L122 (Ethox Chemicals,Greenville, S.C.). After thorough mixing the material was opticallyisotropic and of high viscosity. Of this, 4.609 gm of cubic phase wascombined in a 50 mL beaker with 13.399 gm of a diethanolamine-NATsolution; the latter prepared by mixing 3.746 gm of diethanolamine(Aldrich Chemical Company, Milwaukee, Wis.), 6.746 gm of distilledwater, and 4.495 gm of N-acetyl-DL-tryptophan (MP Biomedicals, Aurora,Ohio). The cubic phase/diethanolamine-NAT mixture was dispersed with aHomogenizer (Brinkmann Polytron PT3000) at 15 k rpm for five minutes.The homogenizer speed was then reduced to 5 k rpm and to it was added1.001 gm of 1% Fluorescent-Labeled Albumin (FITC-albumin, Sigma ChemicalCompany, St. Louis, Mo.) and homogenizing continued for two minutes.After the sample was allowed to sit undisturbed for 20 minutes, 9.100 gmof a 20% wt/wt zinc acetate solution was slowly added while stirring ona stir plate. When magnetic stirring became impossible due to increasedviscosity, the dispersion was hand-stirred with a spatula. The pH wasmeasured to be 8.2 (Hanna Instruments, Woonsocket, R.I.). The sample wasdivided into two test tubes and centrifuged (Clay Adams compactphysicians centrifuge) for 60 minutes at 4800 rpm, and the top, liquidphase, representing the exterior phase to the particles, was examined todetermine whether the fluorescent protein had been taken up by theparticles. To facilitate this, a control sample was prepared by mixingthe same total amount of FITC-albumin into the same amount of water asin the dispersion.

A black light photograph of an aliquot of the top phase (in a glasspipette) and an aliquot of the fluorescent control was taken (notshown). The photograph clearly showed that very little fluorescence,indeed an undetectable amount by eye, was visible in the left pipettewhilst the control on the right was strongly fluorescent yellow-green. Aschematic representation of the experiment and these results are shownin FIG. 6. This Example demonstrates that a protein has been taken up bycubic phase particles prior to coating, after which a solid coating wasapplied by simple addition of zinc ions, and the coated particles easilycollected. As reported in Example 2 above, zinc-NAT coated particlessuch as these do in fact bind to certain selected substrata.

Example 13

In this Example, cubic phase particles were coated with anenergy-absorbing matrix material useable in MALDI and SELDI. The sameprotocol for making zinc-NAT coated particles was used in thisexperiment, except that the N-acetyltryptophan was replaced by anequimolar amount of Trans-3-indolacrylic acid. The resulting particleswere examined by differential interference contrast microscopy and foundto be coated by the energy-absorbing matrix material.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

We claim:
 1. A method of preventing non-specific binding during an assaythat uses a solid support or substrate, comprising the steps of: bindingone or more capture molecules to a surface of said support or substrate;binding lyotropic liquid or liquid crystal material to said surface ofsaid support or substrate at locations on said surface where capturemolecules are not bound, whereby samples exposed to said support orsubstrate are presented with one or more regions for enabling specificbinding said one or more capture molecules and are blocked fromnon-specific binding to said surface of said support or substrate bysaid lyotropic liquid or liquid crystal material.
 2. The method of claim1 wherein said step of binding lyotropic liquid or liquid crystalmaterial is performed by depositing said lyotropic liquid or liquidcrystal material over said surface of said support or substrate aftersaid step of binding one or more capture molecules to said surface ofsaid support or substrate.
 3. The method of claim 1 wherein saidlyotropic or liquid crystalline material is bound to said support orsubstrate in the form of a plurality of particles.
 4. The method ofclaim 3 wherein said particles are coated.
 5. The method of claim 3wherein said particles are uncoated.
 6. The method of claim 1 whereinsaid step of binding said lyotropic or liquid crystalline material tosaid support or substrate is performed by bonding directly to saidsupport or substrate.
 7. The method of claim 6 wherein said bonding ishydrogen bonding.
 8. The method of claim 6 wherein said bonding is ionicbonding.
 9. The method of claim 1, wherein said lyotropic liquid orliquid crystalline material is cubic phase.
 10. The method of claim 3wherein some or all of said plurality of particles are charged.
 11. Themethod of claim 10 wherein said binding step is achieved using a chargeon said particles that are charged to interact with said surface of saidsupport or substrate.
 12. The method of claim 5 wherein some or all ofsaid plurality of particles are charged.
 13. The method of claim 12wherein said binding step is achieved using a charge on said particlesthat are charged to interact with said surface of said support orsubstrate.